Health Test for a Broad Spectrum of Health Problems

ABSTRACT

Provided herein are methods and devices for the detection of conditions or disorders by detecting altered levels of stress response pathway biomarkers. Also provided are methods and reagents for identifying panels of biomarkers associated with a condition or disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/122,130 filed Jun. 17, 2011, now issued as U.S. Pat. No.8,771,962; which is a 35 USC §371 National Stage application ofInternational Application No. PCT/US2009/059438 filed Oct. 2, 2009;which claims the benefit under 35 USC §119(e) to U.S. Application Ser.No. 61/102,341 Oct. 2, 2008, now expired The disclosure of each of theprior applications is considered part of and is incorporated byreference in the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a device for detecting astress response in a sample, and more specifically to methods ofdetecting a stress biomarker in a sample.

2. Background Information

Wellness products and services are increasingly popular, particularly indeveloped countries. Many are rooted in the traditional medicine, e.g.,body care products, natural medicines, massage, acupuncture, sauna, spatreatments. New products and services include handheld devices for vitalsign measurements, tests for ‘good’ or ‘bad’ metabolites such asantioxidants or cholesterol, and treatments such as cold laser orhyperbaric oxygen. Wellness products and services are sold at retailstores, walk-in clinics, integrative medicine facilities, health spasand gyms and through electronic healthcare companies that also provideintegrated wellness services. Wellness products and services aretypically selected using generalized recommendations (e.g., age andgender based), subjective tests such as health questionnaires and thepain scale, vital signs (blood pressure), weight and metabolite tests(e.g., cholesterol or glucose). New test are needed for personalizedassessment of health, identification of the need for wellness productsand selecting the best match for the personal need. Optimally, new testswill provide an early warning of a health problem and indicate thenature of the problem. In addition, new tests showing specific healthbenefits of wellness products and services are needed, in addition tothe available anti-oxidant tests for nutritional supplements.

There is an urgent need for a new health test suitable for point-of-care(POC) settings. The test should be noninvasive and capable of detectingearly signs of deteriorating health status. To be POC-expedient, thetest should be rapid, technically simple and inexpensive.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided POC devicesuseful for Stress Response Profiling (SRP) in saliva. In one embodiment,the device is a hand-held digital device for mobile health monitoring(FIG. 6). The use of the device is intuitive (similar to a digitalthermometer) and does not require training for use. The test isnoninvasive, rapid and inexpensive, and capable of detecting earlywarnings of health problems. The SRP device can be used for preventativePOC health screening, consumer-centric wellness care, routine clinicalcare, it can be used in emergency rooms and trauma units, it can be usedby first responders, by individuals for chronic disease management, itcan be used in complementary and alternative medicine, militaryhealthcare.

In accordance with the present invention, there are provided devices fordetecting at least one stress response biomarker in a test sample. Suchdevices include a disposable module for uptake of a test sample andreagent storage, wherein the module contains reagents for assaying forat least one stress response biomarker; and a reusable module for signaldetection and result display; wherein the reusable module displays asignal that indicates the presence of the at least one stress responsebiomarker in the test sample. In some embodiments, the signal provides adigital readout of a percentage above a baseline representing thepresence of the at least one stress response biomarker in the testsample. In certain embodiments, the signal provides a visual indicationrepresenting the presence of the at least one stress response biomarkerin the test sample. In one aspect, the visual indication is a colorindication.

The device may be used to test a biological sample. In some embodiments,the test sample is selected from the group containing breath air,saliva, urine, sweat, tears, blood, serum, stool, phlegm, bone marrow,cerebrospinal fluid, seminal fluid, vaginal fluid, amniotic fluid, skin,breast milk, tissue, plant sap, an egg, microbial body, cells suspensionor a combination thereof.

In certain embodiments, the device further includes a means foraccessing a database, wherein the database provides a correlationbetween the presence of the at least one stress biomarker molecule inthe test sample and (i) the presence, absence, or severity, if present,of a particular disease state; or (ii) the likelihood that an organismfrom which the test sample was obtained will contract or be subject to aparticular disease state.

In some embodiments, the device assays for at least one stress responsebiomarker selected from the group consisting of aldose reductase,apoptosis signal-regulating kinase 1, aquaporin 5, beta-endorphin,betaine GABA transporter, caspase recruitment domain protein 9, caspase8, cyclin D, cyclooxygenase 2, cytochrome P450, cytochrome c, c-fos,c-jun, epidermal growth factor receptor, ferritin, glucocorticoidreceptor, glucose regulated protein 58, glucose regulated protein 75,glutathione S-transferase p, GroEL, heat shock protein 25/27, heat shockprotein 40, heat shock protein 60, heat shock protein 70, heat shockprotein 90, heat shock transcription factor-1, heme oxygenase-1,interleukin 1β, interleukin 6, interleukin 8, interleukin 10,interleukin 12, laminin, leptin receptor, matrix metalloproteinase 9,metallothionein, Mek-1, Mekk-1, inducible nitric oxide synthase,peripheral benzodiazepine receptor, p38 MAPK, salivary alpha amylase,SAPK, serotonin, serotonin receptor, substance P, superoxide dismutaseMn, superoxide dismutase Cu/Zn, superoxide dismutase EC, transforminggrowth factor β, tumor suppressor p53, and vasoactive intestinalpeptide. In one aspect, the device includes at least one stressbiomarker associated with dehydration. In another aspect, the deviceincludes at least one stress biomarker associated with AIDS progression.

In another embodiment of the invention, there are provided methods fordetecting a condition or disorder associated with a stress response in asubject. The methods include detecting an altered level of at least onebiomarkers in an stress response biomarker panel in a sample comprisingsalivary cells from a subject, as compared to a corresponding samplefrom a normal subject, wherein the panel comprises at least twobiomarkers, and wherein further an alteration in the level of biomarkeris indicative of a stress response associated with the condition ordisorder, thereby detecting the condition or disorder in the subject. Inparticular embodiments, the at least one stress response biomarker isselected from the group consisting of aldose reductase, apoptosissignal-regulating kinase 1, aquaporin 5, beta-endorphin, betaine GABAtransporter, caspase recruitment domain protein 9, caspase 8, cyclin D,cyclooxygenase 2, cytochrome P450, cytochrome c, c-fos, c-jun, epidermalgrowth factor receptor, ferritin, glucocorticoid receptor, glucoseregulated protein 58, glucose regulated protein 75, glutathioneS-transferase p, GroEL, heat shock protein 25/27, heat shock protein 40,heat shock protein 60, heat shock protein 70, heat shock protein 90,heat shock transcription factor-1, heme oxygenase-1, interleukin 1β,interleukin 6, interleukin 8, interleukin 10, interleukin 12, laminin,leptin receptor, matrix metalloproteinase 9, metallothionein, Mek-1,Mekk-1, inducible nitric oxide synthase, peripheral benzodiazepinereceptor, p38 MAPK, salivary alpha amylase, SAPK, serotonin, serotoninreceptor, substance P, superoxide dismutase Mn, superoxide dismutaseCu/Zn, superoxide dismutase EC, transforming growth factor β, tumorsuppressor p53, and vasoactive intestinal peptide. In one aspect, themethod includes at least one stress biomarker associated withdehydration. In another aspect, the method includes at least one stressbiomarker associated with AIDS progression.

In some embodiments, the levels of the at least one biomarker aredetected by analysis of biomarker protein or nucleic acid in the samplecomprising the salivary cells. In particular embodiments, the analysisof biomarker protein includes detection with an antibody. In one aspect,the salivary cells are lysed prior to analysis with the antibody. Theanalysis may be conducted by ELISA or other antibody detection methodsknown in the art. In certain embodiments, the levels of the at least onebiomarker are assayed using a device of the invention. In someembodiments, the sample containing the salivary cells is analyzed onmicroscope slide.

In other embodiments, the analysis of biomarker nucleic acid comprisesisolation of salivary cell nucleic acid. In one aspect, the biomarkernucleic acid is detected in the isolated salivary cell nucleic acid bynucleic acid hybridization or PCR amplification.

In another embodiment of the invention there are provided methods ofprocessing a salivary cell sample for biomarker analysis. Such methodsinclude applying a sample of saliva or salivary cells to a substrate;fixing the cells; incubating the cells in low pH citrate buffer at 37°C.; contacting the cells with serum; applying a primary antibody foreach of biomarker of a biomarker panel; and detecting the binding of theprimary antibody using a secondary antibody having a detectable label,wherein the label is detected optically using a computerized imageanalysis. In certain embodiments, the salivary cells are collected usingan oral brush. In some embodiments, the biomarker panel comprises atleast one biomarker selected from the group consisting of aldosereductase, apoptosis signal-regulating kinase 1, aquaporin 5,beta-endorphin, betaine GABA transporter, caspase recruitment domainprotein 9, caspase 8, cyclin D, cyclooxygenase 2, cytochrome P450,cytochrome c, c-fos, c-jun, epidermal growth factor receptor, ferritin,glucocorticoid receptor, glucose regulated protein 58, glucose regulatedprotein 75, glutathione S-transferase p, GroEL, heat shock protein25/27, heat shock protein 40, heat shock protein 60, heat shock protein70, heat shock protein 90, heat shock transcription factor-1, hemeoxygenase-1, interleukin 1β, interleukin 6, interleukin 8, interleukin10, interleukin 12, laminin, leptin receptor, matrix metalloproteinase9, metallothionein, Mek-1, Mekk-1, inducible nitric oxide synthase,peripheral benzodiazepine receptor, p38 MAPK, salivary alpha amylase,SAPK, serotonin, serotonin receptor, substance P, superoxide dismutaseMn, superoxide dismutase Cu/Zn, superoxide dismutase EC, transforminggrowth factor β, tumor suppressor p53, and vasoactive intestinalpeptide.

In another embodiment of the invention, there are provided methods forconstructing a biomarker panel for detecting a stress response in acultured cell. The method includes detecting the level of one or morebiomarkers from a panel of biomarkers in cultured cells subjected to atreatment that induces cellular stress; and comparing the level of thebiomarkers from the treated cells to the level of the biomarker from acorresponding sample of cultured cells that have not been subjected tothe treatment that induces cellular stress, wherein biomarkers having adifference level in the treated cells as compared to the untreated cellsare included in an SR biomarker panel for a stress response. Inparticular embodiments, the treatment that induces cellular stress is astressor selected from the group consisting of heat shock, freeze/thawcycling, hypersalinity, dehydration, and oxidative stress. In someembodiments, the cultured cells are salivary cells, peripheral bloodmononuclear cells, or cells from organ cultures of tonsil, skin, gut orlung. In particular embodiments, the cells are animal cells. In oneaspect, the cells are human cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of device for collecting saliva samples.

FIG. 2 shows a plot of the levels of stress response (SR) biomarkers insalivary cells treated with different environmental stressors. SRbiomarkers were detected using enzymatic immunochemical staining assaywith mouse and rabbit antibodies and a permanent color label. Thestaining intensity was measured in relative optical density units usingimage analysis. The y axis shows the average staining intensity for 40SR biomarkers. Error bars show standard deviations of the averagestaining intensity. N pool, untreated salivary cells from three donors.Cultured cells from the N pool were treated by desiccation (T1),hypersalinity (T2) or heat shock (T3). S pool, cells combined aftertreatment of the N pool by desiccation, hypersalinity, heat shock,oxidative stress and freeze/thaw shock.

FIG. 3 shows cellular stress in saliva induced by treatment in vitro orby physiological stress in vivo. 40 SR biomarkers were detected insalivary cells using immunochemical staining with a color label. In cellimages, the color is shown as stippling. a, N pool, normal saliva from 3donors. b, S pool, cells combined after treatment by desiccation,hypersalinity, heat shock, oxidative stress and freeze/thaw shock. c,salivary cells from a healthy donor. d, saliva from the same donorduring post-traumatic physiological stress. EMA, positive controlstaining of a cytoplasmic protein in salivary epithelial cells. NC, anegative staining control.

FIG. 4 shows a plot of shows levels of individual SR biomarkers beforeand after treatment with environmental stressors. 52 SR biomarkers weremeasured using immunochemical staining and image analysis. The y axisshows the average staining intensity for the 52 biomarkers. N pool,untreated salivary cells from three donors. S pool, cells combined aftertreatment by desiccation, hypersalinity, heat shock, oxidative stressand freeze/thaw shock.

FIG. 5 shows expression profiles of SR biomarkers induced by treatmentof salivary cells with different stressors. 52 SR biomarkers weremeasured using immunochemical staining and image analysis. Themeasurements were analyzed using hierarchic clustering to depictrelatedness between profiles. The color scheme indicates biomarkerlevels. The lowest level is white, increased levels are gray to black.Similar profiles are in clusters with short dendrogram branches. N,untreated salivary cells from three donors. S, cells combined aftertreatment by desiccation, hypersalinity, heat shock, oxidative stressand freeze/thaw shock. T1a and T1b, desiccation, two subjects. T2,hypersalinity. T3, heat shock.

FIG. 6 shows an illustration of a rapid, hand-held test device forsaliva biomarkers. The device consists of a disposable cartridge foruptake of saliva sample and reagent storage, and a reusable reader forsignal detection and result display. The result indicates the presenceof a saliva biomarker. The digital display window identifies results asnormal (display reads “OK”), moderately abnormal (display reads“CAUTION”) or highly abnormal (display reads “DANGER”).

FIG. 7 a shows images of multi-SRP staining of saliva cells.

FIG. 7 b shows a plot of the SRP score calculated as the ratio betweenthe average staining intensity across 900 saliva cells, and the maximumstaining intensity value for saliva cells.

DETAILED DESCRIPTION OF THE INVENTION 1. SRP Technology

Stress Response Profiling (SRP) is a recently developed technology thatuses molecular biomarkers for multiparametric measurements ofphysiological stress responses. The SRP measurement serves as a novelvital sign. SRP is applicable to a broad spectrum of health threatsincluding environmental stressors, metabolic stressors, psychologicaltrauma, injuries and diseases. SRP measurements quantify physiologicalstress and also discriminate between different types of healthdisorders. SRP biomarkers monitor ten principal homeostatic processes(Table 1).

TABLE 1 SR biomarkers monitor ten principal cellular stress responses.SR SR biomarkers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 201 + + + + + + + + + + + + 2 + + + + + + + + 3 + + + + + + + +4 + + + + + + 5 + + + + 6 + + + + + + + + + + + + + + + + + + +7 + + + + + + + + + + + + + 8 + + + + + + + + + + + +9 + + + + + + + + + + + + + + + + + 10  + + 21 22 23 24 25 26 27 28 2930 31 32 33 34 35 36 37 38 39 40 1 + + + + + + + + 2 + + 3 + 4 + + + +5 + + + + + + + + + + 6 + + + + + + + + + + + + + + + + + + + +7 + + + + + + + + + + + + 8 + + + + + + + + + + + + + + +9 + + + + + + + + + + + + + + + + + + 10  + + + + + + + + SRP biomarkers1-40: beta-endorphin, caspase 8, cyclin D, Cox-2, CYP450, cytochrome c,EGFR, ferritin, glucocorticoid receptor, Grp58, Grp75, GSTp, Hsp25/27,Hsp40, Hsp60, Hsp70, Hsp90, HSF1, HO-1, IL-1 beta, IL-6, IL-8, IL-10,laminin, leptin receptor, metallothionein, Mekk-1, Mek-1, NADPH-CYP450reductase, iNOS, Fos, Jun, serotonin receptor, serotonin, Substance P,SOD Mn, SOD Cu/Zn, TGFbeta, p53, vasoactive intestinal peptide. SR,stress responses 1-10: redox control, cellular detoxification Phase Iand II, chaperoning, DNA repair, cellular adhesion and motility, cellgrowth and energy metabolism, apoptosis, neuro-endocrine signaling,immunological activation, microbial activation.

As used herein, the term “homeostasis” is a biological process thatmaintains the health of organisms.

As used herein, the term “persistent homeostatic perturbation” is to beunderstood as a homeostatic change that has an adverse effect on thehealth of organisms. It is another way of referring to “chronic stress”or simply “stressed” which should be understood to mean a persistentperturbation of homeostasis and encompassing all forms of chroniccellular stress and chronic physiological stress.

As used herein, the term “stressor” is to be understood as all forms ofagents or conditions that give rise to stress. Stressors according tothe present invention include agents and conditions that are in theouter environment of organisms such as the air temperature as well asagents and conditions that are in the inner environment of organismssuch as a disease.

As used herein, the term “adaptive stress response” or simply “stressresponse” is to be understood as a homeostatic process that provides acountermeasure to stress.

As used herein, the term “stress response pathway” is to be understoodas the form of the stress response that has a specific function in theorganism, such as DNA repair. Stress response pathways are embodied inexpressed molecules (i.e., SR biomarkers.)

As used herein, the term “universal stress response pathway” or simply“SR pathway” is to be understood as a form of stress response to moststressors, in most organisms. Functional activation of these SR pathwaysgenerates reproducible patterns of expressed molecules.

As used herein, the term “SR biomarker” is to be understood as anexpressed molecule known to be or suspected of being associated withactivation of a SR pathway.

As used herein, the term “SR biomarker profile” is a multi-dimensionalpattern of data whose components are at least two SR biomarker scoresfor individual SR biomarkers across a SR biomarker panel.

As used herein, the term “SR pathway profile” is a multi-dimensionalpattern of data representing at least two SR pathways. The componentsare functions of SR biomarker scores related to the individual SRpathways. The functions yield one-dimensional data points that providesimple-to-use indices of activation levels for the individual pathways.

As used herein, the term “stress response profiling” refers toconstructing either or both SR pathway profiles or SR biomarker profilesfrom SR biomarker assays.

As used herein, the term “SR biomarker panel” is to be understood as atleast two SR biomarkers that as a group provide enhanced informationabout stress responses than single SR biomarkers.

As used herein, the term “SR biomarker panel score” or “panel score” isto be understood as a one-dimensional data point calculated as theaverage of SR biomarker scores across a SR biomarker panel.

As used herein, the term “SR biomarker score” is to be understood as anormalized and optionally log-transformed measurement of a SR biomarker.

As used herein, the term “measurement” of a SR biomarker is to beunderstood as a quantitative or qualitative determination of the SRbiomarker's expression level in a sample from an organism.

As used herein, the term “individual SR biomarker assay” or “SRbiomarker assay” is to be understood as an assay of individual SRbiomarkers.

As used herein, the term “combined SR biomarker assay” is to beunderstood as an assay that yields measurements representative of thecombined expression levels for a panel of SR biomarkers.

The homeostatic processes monitored by SRP biomarkers regulate generalstress responses, basic body functions and physical vital signs (bodytemperature, heart rate HR, blood pressure BP, respiratory rate). Thesehomeostatic processes include redox control, cellular detoxification,chaperoning, DNA repair, cellular adhesion and motility, cell growth,apoptosis, neuron-endocrine signaling, immunity, and microbialactivation.

Redox Control (1).

This pathway regulates levels of reactive oxygen and nitrogen species(superoxide, nitric oxide, carbon monoxide) through free radicalscavenging proteins such as superoxide dismutases. Free radicals areessential cellular mediators but when in excess, they cause cellulardysfunction through damaging lipids, proteins, DNA and membraneintegrity.

Cellular Detoxification (2).

Cellular detoxification provides a defense against chemical threats tocellular integrity. Phase I detoxification is a cytochrome P450 drivenprocess for metabolizing a wide variety of endogenous metabolites (e.g.,fatty acids, steroids) and foreign substances (drugs, alcohol,pesticides and hydrocarbons). Phase II is based on the glutathionemetabolism and provides cellular resistance to oxidants, hydrocarbonsand heavy metals.

Chaperoning (3).

Chaperones fold newly synthesized polypeptides and denatured proteinsand for prevent uncontrolled protein aggregation. Chaperoning involveshundreds of “client” proteins and therefore has a key role in multiplebiological functions including cellular protection, metabolism, growth,the development of multicellular organisms and molecular evolution.Excessive chaperoning facilitates disease by folding “wrong” clientssuch as the diphtheria toxin or mutant p53 that are cytotoxic or causecancer.

DNA Repair (4).

DNA damage is ubiquitous and therefore the stability of the genome isunder a continuous surveillance by multiple DNA repair mechanisms. DNAlesions are produced during transcription and replication, and bymetabolic and immunity by-products (e.g., free radicals produced duringaerobic respiration and by immune cells killing bacteria). DNA can bealso damaged by environmental mutagens such as oxidants, heavy metals,radiation and viruses. The DNA repair pathway regulates multiple stagesand mechanisms of DNA repair, and is closely linked with cell cyclecontrol and apoptosis.

Cellular Adhesion and Motility (5).

This pathway monitors cellular interactions with the extracellularmatrix and also changes in cytoskeletal matrix such as centrioles,kinetosomes and other microtubule organizing centers. These processesare essential for cellular survival, growth, metabolism and motility,and also for the formation of microbial biofilms and microbial-hostinteractions.

Cell Growth (6).

In multicellular organisms, cell cycle progression is strongly regulatedduring the development and modulated by growth factors (mitogens),disease and environmental stress. In mature tissues, most cells do notdivide. Cycling cells in tissues are typically somatic stem cellsinvolved in normal tissue turnover (e.g., the germinal layer of theskin). Cell cycling is typically arrested in starved cells and in cellswith DNA or mitochondrial damage. Increased cell growth occurs duringimmune responses, wound healing and regeneration of tissues damaged byenvironmental stress, toxins, disease or infection. Uncontrolled,excessive cell growth is found in cancer.

Cell Death (7).

The programmed cell death (apoptosis) “recycles” cellular components andprevents the release of toxins from dying cells, as happens duringnecrotic cell death. In animal tissues, apoptosis is increased in areasof tissue remodeling and wound healing, and during aging. During adisease, apoptosis can be increased within the diseased tissue (e.g.,psoriatic skin lesions) and/or in remote tissues and biofluids (e.g.,HIV Tat protein is a soluble mediator that triggers apoptosis inuninfected lymphocytes). Apoptosis can be also triggered byenvironmental stressors that cause mitochondrial damage (e.g., oxidativestress and uv light).

Neuro-Endocrine Signaling (8).

This pathway is crucial for regulating physiological homeostasis andbehavioral regulation in animals including simple invertebrates. Itinvolves a large number of mediators (hormones, neuropeptides,neurotransmitters) and cellular receptors produced by specializedtissues (glands and neural tissues), and also locally in peripheraltissues (e.g., skin and gut). In vertebrates, two signaling mechanismsprovide initial responses to stress: the limbichypothalamic-pituitary-adrenal (LHPA) axis that involves glucocorticoids(e.g., Cortisol) and the sympathetic nervous system activation viacatecholamines. However, chronic stress also activates signaling of painand anxiety, energy balance, metabolism, respiration, circulation andreproduction. Neuro-endocrine and immune signaling are integratedthrough common mediators and provide coordinated responses toenvironmental stress and disease.

Immunity (9).

Immunity provides a systemic defense against biological threats toorganism's integrity such as injuries, tumors and disease-causingmicroorganisms. Innate immunity provides a nonspecific defense throughsoluble mediators (e.g., chemokines, agglutinins) and specialized cells(e.g., macrophages) that circulate through the organism and inactivateparasitic microorganisms, engulf apoptotic cell debris and kill infectedand tumor cells. Innate immunity is found in protists, animals andplants. Vertebrates use innate immunity during the initial phases ofstress response because it takes several days to activate specificimmunity that provides threat-specific antibodies and lymphoid cells.Immune regulation is mediated through numerous signaling proteins calledcytokines or interleukins. Increased immunity can be beneficial (e.g.,short-term immune activation that removes a bacterial infection) orharmful (e.g., chronic inflammation and autoimmunity increasephysiological stress through oxidative stress and apoptosis).

Microbial Activation (10).

This pathway monitors the activation of stress responses inmicroorganisms (bacteria, fungi, viruses), and signaling betweenmicroorganisms and host cells. Commensal microbial biofilms are anintegral part of animal and plant bodies and contribute to physiologicalhomeostasis. In animals, microbial biofilms are primarily associatedwith the inner and the outer body surfaces (the mucosal epithelium andthe skin). Therefore microbial biofilms are sensitive both toenvironmental stressors (e.g., uv light) as well as tomicro-environmental conditions in host tissues and body fluids (e.g.,oxidative stress). During physiological stress, increased signalingbetween microbial biofilms and host cells promotes protection of theorganism through modulating host's stress responses. For example,signaling by gastrointestinal microflora modulates levels of proteinswith key roles in redox control, cellular detoxification, chaperoning,cell growth, apoptosis and immunity such as metallothionein, Hsp25,ferritin, p53, TGF beta, IL-8 and IL-10. When pathogenic microorganismsinvade animals or plants, their stress responses are elevated, which inturn increases stress responses in the host (bacterial heat shockproteins are animal superantigens). Disease-causing microorganisms alsorelease soluble mediators that trigger cellular stress and activatemultiple stress response pathways in infected as well as remote hosttissues (e.g., HIV Tat protein).

Stress Response (SR) Biomarkers

Activation of SR pathways by stressors results in a pattern of expressedmolecules such as genes, proteins, metabolites and lipids, referred toherein as “SR biomarkers. Accordingly, each of these biomarkers is saidto be “associated with” one or more SR pathways. Measuring the levels ofthese SR biomarkers provides useful information about the biologicaleffects of stressors. Preferably, the SR biomarkers are expressedmolecules such as proteins or fragments thereof, so long as the fragmentis capable of being recognized in an SR biomarker assay with the samesensitivity as the entire protein.

Preferred SR biomarkers and their known associations with SR pathwaysare listed in Table 2. Additional SR Biomarkers and some but not all oftheir known associations with SR pathways are listed in Table 3.

TABLE 2 SR Biomarkers: Association with SR Pathways and Expression inTaxonomic Groups of Organisms Abbreviated Expression SR pathways # SRbiomarker name A B C D E 1 2 3 4 5 6 7 8 9 10 1 Beta-endorphinEndorphin + + + 0 0 0 0 0 1 0 1 1 0 2 Caspase8 Caspase 8 + + + 0 0 0 1 00 1 0 1 0 3 Cyclin D1 Cyclin + + + 0 0 0 1 0 1 1 0 0 0 4 Cyclooxygenase2 Cox-2 + + 1 0 0 0 0 1 1 1 1 0 5 Cytochrome P 450 CYP450 + + + + + 1 10 0 0 1 1 0 0 0 6 Cytoplasmic cytochrome c Cytc + + 0 0 0 1 0 1 1 0 1 07 Epidermal growth factor receptor EGFR + + + 1 0 0 0 1 1 1 1 1 0 8Ferritin Ferritin + + + + + 1 0 0 0 0 1 1 1 1 1 9 Glucocorticoidreceptor GR + 1 1 0 0 0 1 1 1 1 0 10 Glucose regulated protein Grp58Grp58 + + 1 1 1 0 0 1 0 0 0 0 11 Glucose regulated protein Grp75Grp75 + + + + + 1 0 1 0 0 1 0 1 1 0 12 Glutathione-S-transferase pGST + + + + + 1 1 0 1 0 1 1 0 1 0 13 Heat shock protein 25/27Hsp25/27 + + + + + 1 1 1 0 0 1 1 1 1 1 14 Heat shock protein 40Hsp40 + + + + + 0 0 1 0 0 1 0 1 1 0 15 Heat shock protein 60Hsp60 + + + + + 0 1 1 0 0 1 0 1 1 0 16 Heat shock protein 90Hsp90 + + + + + 1 1 1 1 1 1 1 1 1 0 17 Heat shock transcription factorHSF-1 HSF-1 + + + + + 1 1 1 0 0 1 1 0 1 0 18 Heme oxygenase-1 HO-1 + + 10 0 0 0 1 1 1 1 0 19 Interleukin IL-1beta IL-1 + + 0 0 0 0 1 1 0 1 1 020 Interleukin IL-6 IL-6 + + + 1 0 1 0 0 1 0 0 1 0 21 Interleukin IL-8IL-8 + + 0 0 0 0 1 1 0 1 1 1 22 Interleukin IL-10 IL-10 + 0 0 0 0 1 1 01 1 1 23 Interleukin IL-12 IL-12 + 0 0 0 0 1 1 0 1 1 0 24 LamininLaminin + + 1 1 0 0 1 1 1 1 1 0 25 Leptin receptor ObR + 0 0 0 0 0 1 1 11 0 26 Metallothionein MT + + + + + 1 0 0 1 0 1 1 1 1 27Stress-activated MAP kinase Mekk-1 Mekk-1 + + + + + 0 0 0 0 1 1 0 1 1 28Mitogen activated MAP kinase Mek-1 Mek-1 + + + + + 0 0 0 1 1 1 1 1 0 29NADPH-cytochrome P 450 reductase CYP red + + + + 1 1 0 0 1 1 1 1 0 30Nitric oxide synthase II, inducible iNOS + + + + 1 0 0 0 0 1 1 1 1 1 31Proto-oncogene c-Fos protein Fos + + + 0 0 0 in 1 1 1 1 1 0 32Proto-oncogene c-Jun protein Jun + + + + 0 0 0 in 0 1 1 1 1 0 33Serotonin receptor 5HT R + + + + 1 0 0 0 0 1 1 1 1 0 34 Serotonin5HT + + + + 0 0 0 0 1 1 0 1 1 0 35 Substance P Substance P + + 0 0 0 0 11 0 1 1 0 36 Superoxide dismutase Mn SOD Mn + + + + + 1 0 0 1 1 1 0 1 11 37 Superoxide dismutase Cu/Zn SOD Cu/Zn + + + + + 1 0 0 1 1 1 0 1 1 138 Transforming growth factors beta-1,2,3 TGF + + + + 0 0 0 0 0 1 1 0 11 39 Tumor suppressor p53 p53 + + + 0 0 0 1 0 1 1 0 0 1 40 Vasoactiveintestinal peptide VIP + + 0 0 0 0 0 1 0 1 1 0

TABLE 3 SR Biomarkers: Association with SR Pathways Abbreviated SRpathways # SR biomarker name 1 2 3 4 5 6 7 8 9 10 41 Heat shock protein70 Hsp70 + + + + + 42 Matrix metalloproteinase 9 MMP + + + 43 Aldosereductase ALR + + + + 44 Apoptosis signal-regulating kinase 1 ASK + 45Aquaporin 5 AQP + + + 46 Betaine GABA transporter 1 BGT + + + + + 47SAPK SAPK + + 48 Caspase recruitment domain protein 9 CARD + 49 P38 MAPKp38 + + 50 Peripheral benzodiazepine receptor PBR + + + + + 51 Salivaryalpha-amylase SAA + 52 GroEL GroEL + + 53 Superoxide dismutase EC SODEC + + + 54 Cell adhesion molecules V-CAM, I-CAM + + 55 Monocytechemotactic protein 1 MCP + + 56 Catalase Cat + 57 Hypoxia inducedfactor 1 alpha HIF-1 + 58 Glutathion peroxidase GSHPx + 59 Carbonicanhydrase CAA + + 60 Ornithine decarboxylase ORD + 61 Vasoendothelialgrowth factor VEGF + + 62 Erythropoietin EPO + + + 63 MelatoninMelatonin + + 64 Thyroid-stimulating hormone receptor TSHR + 65Methenyl-tetrahydro-folate reductase MTHFR + 66 Oxytocin Oxytocin + 67Thromboxane synthase 1 TBXAS1 + + + 68 C-reactive protein CRP + 69TNF-alpha TNF + 70 Apolipoproteins A and B apo + + + + 71 Toll-likereceptor TLR + + 72 TspO protein TspO + 73 Bacterial trehalose synthaseTre-6P + + 74 Bacterial Sigma S factor RpoS + 75 Protease DegP DegP + 76Superoxide dismutase Fe SOD Fe + + + 77 Glutathione reductase A gorA + +78 Ferric uptake regulator fur + + 79 Multidrug efflux pump acf + + 80Sigma-B factor Sigma B + 81 DNA-binding protein stationary phase dps +82 DnaJ DnaJ + + 83 GroES GroES + + 84 8-hydroxy-deoxyguanosine8-OH-dG + 85 8-hydroxy-guanine 8-OH-G + 86 DNA damage binding protein-2DDB2 + 87 Xeroderma pigmentosum (XP) C protein XPC + 88 DNA qlycosylaseOGG1 OGG1 + 89 pyrimidine-base DNA glycosylases NEIL + 90 uracil DNAglycosyiase UNG + 91 thymidine DNA glycosyiase TDG + 92 DNA glycosylaseMTH1 + 93 Apurinic/Apyrimidinic endonuclease APE + 94 MSH-2 MSH-2 + + +95 MLH-1 MLH-1 + + + 96 Senescence-associated beta-qalactosidaseSA-beta-gal + + + 97 P21 p21 + + +

The relationship between each individual stressor and the ten SRpathways, and thus the SR biomarkers associated therewith, may notalways be known, especially since the effects of many stressors onparticular SR pathways is not yet well studied. For example, the effectsof bird flu virus, engineered nanoparticles, and effects of deep spaceand deep sea or other extreme environments on each individual SR pathwaymay not be completely elucidated.

However, most SR biomarkers associated with the 10 SR pathways areuseful targets in assays to analyze the effects of both known andunknown stressors, such as environmental stressors and/ordiseases-related stressors. Accordingly, SR biomarkers associated withSR pathways are suitable targets for studying the effects of unknownstressors because they provide a response-oriented detection strategythat does not require prior knowledge of the stressor.

SR Biomarkers associated with the SR pathways are also suitable targetsin studying the effects of complex stressors, some of which may be knownand others of which may be unknown. These complex, or “combined”stressors, are common in real-life scenarios, and may include multipleknown and unknown adverse conditions. Global warming, ozone holes, humaneffects on wildlife, urban pollution, natural and industrial disasters,poverty and war are examples of complex, combined stressors.

2. Dehydration

Dehydration is a water and electrolyte disorder that can severely affecthuman performance and health¹⁸⁻²¹. Preventable dehydration affects over90 million people in the US and the costs exceed 10 billion annually forunnecessary hospitalizations and avoidable complications¹⁸⁻⁵⁵. 3%dehydration (i.e., the loss of 3% total body water) is a criticalend-point for dehydration diagnostics in field settings because it hasmeasurable negative health consequences but it can be simply treated byoral rehydration^(19,21,26,38,56-57). 3% dehydration can be caused bysweating during strenuous physical work with restricted fluid intake orby extremes of temperature, humidity or altitude^(19,21,29-30,58). Thistype of dehydration frequently affects soldiers, athletes, constructionworkers, policemen and firefighters^(21,24,26,28,30,44,53,59-63). Acutedehydration due to gastroenteritis is frequent inchildren^(34-36,39-42,46). Chronic dehydration associated with oraldisease is a common side-effect of cancer therapy or diuretics indiabetic and dialysis patients^(31-33,45). Elderly are at a greater riskfor dehydration because the mechanism controlling thirst becomes lesssensitive with age, and dehydration occurs more rapidly due to a lowerwater content in the aging body^(25,47,49,51,64). Many patients in theterminal phase of their illness experience dehydration due to a varietyof causes related to their disease or treatment^(32-33,48). Dehydrationis a major cause of mental deterioration and death in patients withAlzheimer's disease^(25,64). Life-threatening complications ofdehydration include heat stroke, heat illness and hyponatremia due toover-aggressive rehydration^(19-21,27-28,30,44,53,61,65).

Dehydration and hyponatremia are also common in patients with cysticfibrosis, kidney, heart and liver disease. Currently used methods fordetecting dehydration are based on laboratory analysis of blood, urine,saliva and anthropometric indices such as body mass measurement. Thegold standards for dehydration assessment are blood osmolality, bodymass loss and TBW measurement using isotope dilution. However, nodehydration test is currently available for wellness and diseasemanagement in POC settings and in field conditions. A field-expedientdehydration test is also needed for monitoring the performance andhealth of military service members during training and deployment. Thus,there is an unmet need for non-invasive, rapid and accurate test for ≧3%dehydration that could be administered frequently to monitor thehydration status of at-risk individuals in fieldsettings^(18-19,24,44,59).

The dehydration test provided herein based on salivary SR biomarkersrisk is radically different from current dehydration tests because itutilizes a new assay principle based on based on monitoring thephysiological status of the patient. Recent studies indicate that thehomeostatic processes monitored by SRP are also activated bydehydration. As shown in Table 4, SRP biomarkers are relevant to thesensing of water loss.

The assay principle was reduced to practice using an immunoassay of SRbiomarkers that monitor the physiological status based on cellularstress responses in saliva. The saliva test is noninvasive, rapid andinexpensive. In contrast, the current dehydration tests monitor physicalproperties (e.g., osmolality) using blood or urine samples andexpensive, time consuming laboratory assays.

TABLE 4 SRP Biomarkers and Molecular Sensing of Dehydration Cellular andmolecular effects of dehydration Homeostatic process SRP Efflux ofintracellular water Chaperoning + Increased intracellular salinity Redoxcontrol + Cell membrane distortion Cell adhesion and motility +Macromolecular crowding and Chaperoning + denaturation Increasedxenobiotics production Cellular detoxification + Oxidative stress Redoxcontrol + Cell growth arrest Cell growth + Pro-apoptotic signalingApoptosis + Hormonal changes Neuro-endocrine signaling + InflammationImmunity + Microbial biofilm changes Microbial activation +

3. Occupational Stress

Some occupations involve exposures to complex environmental andpsychological stressors, for example astronauts, pilots, divers,soldiers, police and haz-mat personnel. These occupational stressors aretypically much more diverse and higher, compared to mainstreamprofessions. Occupational stress can have adverse effects on health andperformance and therefore needs to be monitored. Currently, there are notests for occupational stress.

Occupational stressors can be physical (radiation, health, cold,altitude, gravity, vibrations), chemical (low air oxygen, toxicchemicals, micronutrient deficiency), biological (pathogens, injuries,jet leg, sleep deprivation) or psychological. Often, they involveundefined factors (e.g., outer space radiation, new pathogens).

There are two basic strategies for the detection of occupationalstressors. The first one measures levels of potential health threats inthe environment (e.g., levels of toxic chemicals). This approach is notsuitable for synergistic stressors (e.g., a mixture of individually safechemicals can be toxic), undefined agents (e.g., new types of pathogensor space radiation) and psychological stressors. The second strategy isbased on measuring changes in health status during and after exposures.Currently used health monitors measure vital signs and perform metabolicblood/urine tests. These monitors are not sensitive to many occupationalstressors and often they are invasive and not practical (e.g., a nest ofwires for vital signs). New health tests under development measuremolecular indicators of immunological status (e.g., cytokines and latentviruses in saliva and blood) or assess cognitive performance (e.g.,specialized computer games).

The device of the invention offers a better solution for the assessmentof complex occupational stressors than any of the current methods. Themain advantages of SRP are broad-based sensitivity and new insights intothe mechanism of stress. The SRP sensitivity allows an upstream, earlydetection strategy for a broad spectrum of occupational stressors. Incontrast, current methods provide a downstream detection strategyfocused on delayed effects. For example, SRP can measure moleculareffects directly triggered by a radiation exposure such as increasedfree radical levels and protein denaturation. These effects precedeimmunological or cognitive changes by several hours to several days. Theinsights into the mechanism of occupational stress could be used for thedevelopment of countermeasures.

4. Male Fertility Test

Current male fertility tests use sperm counts and blood and salivaassays for reproductive hormones. Semen is a complex mucosal fluid withmultiple cell types similar to saliva and milk. The device of theinvention can be used to measure cellular stress in semen as anindicator of sperm health and a predictor of male fertility.

5. Embryonic Health Test

Current prenatal health tests use amniotic cultures to perform FISHassays for genetic abnormalities. SRP-based cellular stress test of theamniotic culture could serve as a new indicator of embryonic health anda predictor of prenatal health.

6. Oral Health Test

Current oral health tests use X rays and dental exams to detectperiodontal disease, a serious chronic health problem. New expensivegenetics tests are under development. SRP-based cellular stress insaliva could serve as a new indicator of oral health and a predictor ofperiodontal disease.

7. Saliva as a Diagnostics Sample

Oral diagnostics is a rapidly growing field that provides a convenientalternative to blood sampling for a rapidly expanding list of analytesand diseases, including an early test for a heart attack. It haspreviously been reported that SRP biomarkers in saliva were sensitive tochronic diseases and post-traumatic psychological stress. Saliva can besampled simply and noninvasively. A typical saliva sample (10 drops,about 0.3 ml) contains about a half million of epithelial and whiteblood cells. Salivary cells may play a role in the cellular andmolecular mechanism of oral disease transmission. It was also found thatsalivary cells express SRP biomarkers and that salivary SRP levels arestrongly increased by stress.

Saliva diagnostics provides a noninvasive and safe alternative to bloodor urine diagnostics¹⁻³. The main challenge of using saliva as adiagnostic sample is the inherently low concentration of solublebiomarkers in the cell-free saliva fluid¹⁻³, which is the currently usedmethod for saliva sampling¹⁻³. We found that saliva contains a largenumber of cells (about 10⁶/ml) that contain clinically significantbiomarkers⁴⁻⁷. Therefore, saliva samples that contain salivary cellsprovide a radically improved diagnostic sample for saliva diagnostics.

Saliva samples may be collected by the subject or may be collected by apassive method in which a health care worker or person other than thesubject collects the sample. For example, unstimulated saliva samplesmaybe be collected having the subject collect saliva into a sterilecontainer. Alternatively, saliva samples may be collected using a devicesuch as a small oral brush by brushing teeth and gum surfaces on bothsides of the mouth for about 20 seconds. To collect a larger volume ofsaliva, the brushing may be repeated using additional brushes. Acomparison of saliva samples collected using self-brushing, and brushingperformed by an assistant, showed that both methods yielded the sameaverage sample volume and cellular composition. The passive salivasampling method (brushing performed by an assistant) enables salivadiagnostics in subjects that cannot actively participate in activesaliva sampling such as infants, elderly, unconscious patients ormentally ill patients. Currently, no other passive saliva collectionmethods were published that enable saliva diagnostics in these subjects.

The device for collecting salivary cells may consist of a disposablebrush and a reusable handle. The brush is suitable for oral use. Thebrush may be used to collect saliva by brushing gums and teeth, and thenmay be used to process the saliva into smears on slides or lysates.

Methods that collect saliva samples containing salivary cells provide aradically improved diagnostic sample for saliva diagnostics becausesalivary cells contain clinically significant biomarkers (e.g., proteinsand DNA)⁴⁻⁷. In contrast, current methods for saliva sampling collectcell-free saliva that has inherently low concentration of solublebiomarkers, which is the main challenge in using saliva as a diagnosticsample¹⁻³.

In one embodiment of the invention, there are provided referencereagents and materials for saliva diagnostics generated by inducingcellular stress in cultured normal salivary cells by in vitro treatment.Although as described below, the induction of proteomic biomarkers using5 stressors is exemplified, the treatment principles could easily beadapted to protocols that use different stressors, or other stressmarkers such as mRNA, DNA, reporters or small molecules associated withthe activation of SR pathways (see Table 1). Moreover, the methodprinciples could be also easily adapted for other human cells that havea diagnostic value, e.g., white blood cells, and for diagnostic cellsfrom animals, plants and microorganisms. Although the production of cellsmears as reference materials is exemplified, the method principlescould easily be adapted to the production of other types of referencematerials and reagents such as protein lysates, mRNA lysates orcell-free saliva fluid.

Methods that induce cellular stress in cultured normal salivary cells byin vitro treatment provided novel reference reagents materials forsaliva diagnostics such as salivary cell smears with normal andincreased levels of salivary biomarkers. The in vitro production ofreference reagents and materials was rapid, convenient and inexpensive.The new reference materials are radically different from currentreference materials produced using cell-free saliva samples¹⁻³ collectedfrom patients with and without specific medical conditions.

Also provided herein are methods for the development of salivarybiomarker assays. In one example, the method uses reference reagents andmaterials prepared as described in Example 2. Two types of laboratorysaliva immunoassays are exemplified, the immunocytochemical (ICC) assayand the ELISA assay. Together, these assays enable the development andvalidation of saliva biomarker panels because they provide complementaryanalysis of cell-associated (ICC) and soluble (ELISA) saliva biomarkersas described in Example 5. In addition, laboratory saliva ELISA assaysare also useful as reference assays for the development of commercialsaliva assays such as the lateral-flow immunoassay (LFIA) test strip.Although as described below, the development of saliva ICC and ELISAassays exemplified, the principles of the method could easily be adaptedto developing other types of immunoassays such as LFIA, and to assaysthat measure other types of markers such as mRNA, DNA or smallmolecules. The method for developing new saliva assays using referencereagents and materials produced in vitro was rapid, convenient andinexpensive compared to currently used methods based on clinical salivasamples. Saliva assays produced by the new method use saliva samplesthat contain salivary cells and therefore the new assays are radicallydifferent from current saliva assays based on cell-free salivasamples¹⁻³.

Also provided are methods for measuring baseline concentrations ofsaliva biomarkers using two complementary assays, ICC forcell-associated biomarkers in saliva smears, and ELISA for solublebiomarkers in saliva lysates. In one example, the method uses referencereagents and materials prepared in vitro as described in Example 2.Biomarker baselines in normal saliva provide a useful benchmark theconstruction of biomarker panels for saliva diagnostics as described inExample 5. Although as described below, the baseline measurement of aprotein biomarker using the ICC and ELISA assays is exemplified, theassay principles could easily be adapted to measure soluble proteinmarkers using other assays such as the lateral-flow immunoassay, andother types of biomarkers such as mRNA, DNA or small molecules.

Also provided are methods for constructing a biomarker panel that isuseful for salivary diagnostics of health disorders. Although asdescribed below, panels of proteomic markers are exemplified, the sameprinciple could easily be adapted to measure, for example, mRNA, DNA orsmall molecules, and to assess the diagnostic value of the salivabiomarker panel using other statistical methods than exemplified here.The new method is rapid, convenient and inexpensive because it usesreference reagents produced in vitro, compared to current methods thatuse reference reagents based on clinical saliva samples¹⁻³.

8. LFIA Biosensors

The first FDA-approved commercial oral biosensor broke the ground for asuccessful commercialization of oral diagnostics. This breakthrough testis ORAQUICK ADVANCE® RAPID HIV-1/2 ANTIBODY TEST produced by OraSure.The test is based on a mature biosensor technology, the lateral-flowimmunoassay (LFIA). LFIA is rapidly expanding to provide a wide varietyof POC and point-of-need tests for wellness (pregnancy and ovulationtests), public health (HIV and Hepatitis C tests) and forensics (drugand alcohol tests).

The present invention provides a simple analyzer that provides aquantitative measurement of physiological stress. Examples of theanalyzer is shown in FIGS. 6 and 7. The analyzer consists of a reusablereader with a digital result display and disposable cartridges (teststrips). Alternatively, the whole unit is disposable. The test result isa “stress number,” or “SRP score,” which is a quantitative indicator ofthe general stress level. A color-coded results window is used forsimplified interpretation of the stress number: e.g., green for normal,yellow for mild/moderate and red for high stress levels (similar to adigital thermometer). If the stress number is above the normal range, adigital message prompts the user to obtain advanced analysis of theirstress, and to seek medical advice.

In one embodiment of the analyzer device, there is provided a pen-sizedigital device consisting of a disposable teststrip, a reusableelectronic reader and a clip-on holster. The user briefly puts the endof the test strip in the mouth to collect saliva and then inserts thetest strip into the reader. In less than 3 minutes, the reader shows adigital stress measurement. A color guide shows whether results indicatenormal health (green), a mild health problem (yellow) or serious healthdeterioration (red). Results from multiple tests are stored on board andcan be wirelessly communicated to a remote care center. The deviceoperates autonomously using small batteries that last for several weeks.

An advanced analyzer that provides quantitative and qualitativemeasurements of physiological stress is also provided in the presentinvention. The analyzer uses a disposable cartridge to perform a highlymultiplexed immunoassay (10-40 individual biomarkers). Assay results aremeasured and processed using an opto-electronic reader. Test results aredisplayed digitally. The analyzer is equipped for data transfer (USB,bluetooth) to a web-based service (StressNet) that supports advancedanalysis of test results. The analyzer can be a desktop device or aPDA-class handheld device.

The average SRP score is computed by the device based on measurements ofthe individual biomarkers and displayed in the results window at the endof the test. The average SRP score will be interpreted as a quantitativeindicator of the general stress level, analogous to the “stress number”(combined SR biomarker score), since the average SRP score stronglycorrelated with combined SR biomarker scores in reference samples. Ifthe general stress level is above the normal range, the user will beprompted to perform advanced analysis of his SRP profile using a webservice. The software on the web service interprets the SRP profile andprovides user-friendly results in the form of a short message. Themessage (1) describes the nature of personal stress, e.g., the % risk ofspecific health problem, (2) shows top three personal stress drivers,(3) recommends wellness products/services that are the best match forthe personal stress drivers, and (4) directs the user to additionalinformation.

9. Predictive Medical Diagnostics and Personalized Disease Management

Many health problems and diseases do not have a lab test that couldprovide early warnings before the onset of clinical symptoms. This isparticularly true for mental diseases. Often, the earlier a disease isdiagnosed, the more likely it is that it can be cured or successfullymanaged. Managing a disease, especially early in its course, may lowerits impact on life or prevent or delay serious complications. Diseasemanagement strategy strongly depends on the ability to predict theseverity of a disease, for example, differentiating metastatic cancer,Alzheimer's dementia, or kidney failure. Such predictive tests arecurrently unavailable.

Chronic diseases are associated with early physiological changes thatmight be detectable using SRP biomarkers in saliva. Early disease testscan be done based on stress-induced changes in microflora and mobilegenetic elements (MGE). SRP profiles might reflect the course of adisease. For example, a metastatic cancer might have a different SRPprofile than a non-metastatic cancer because metastatic cells producechemical compounds and biological interactions that are likely to affectstress responses in the normal cells and biofluids analyzed in SRPassays.

Methods: The SRP panel (40 or 41 biomarkers) is analyzed in samples fromcase/control subjects (cases represent a disease, e.g., breast cancer,BC). If SRP profiles unambiguously discriminate between BC and controls,the panel is minimized toward the smallest panel sufficient for the BCclassification. Based on the preliminary data, the minimized panel willconsist of 3-6 biomarkers. If SRP profiles from the original SRP paneldo not discriminate BC, new biomarkers are added to the panel until theSRP profile can discriminate BC. The new biomarkers are selected usingthe SR pathway profile of BC determined by the original SRP panel. Forexample, if BC-related stress preferentially involves misfoldedproteins, oxidative stress and changes in cell cycle and growth, thennew biomarkers for these processes will be preferred. When SRP profilecan discriminate BC, the biomarker panel will be minimized as describedabove. The minimized panel is detected using combined SR biomarkers(“multi-SRP assay’), if a combined score is sufficient for BC detection.Alternatively, the biomarkers in the minimized panel are measuredindividually if a SRP profile is needed for BC detection. A multi-SRPtest or an assay for 3-6 individual SRP biomarkers will be implementedusing the device of the invention.

10. Human Disease Research

The cellular and molecular mechanism of a disease (i.e., molecularpathogenesis) shows where and how the disease harms the body. Diseasesperturb cellular and physiological homeostasis, and the perturbationpattern might be disease-specific and reproducible (i.e., adisease-specific stress signature). Disease countermeasures prevent,reduce or remove the disease-specific stress signature. Disease-specificstress signatures can be analyzed by SRP. This information can be usedto guide the development of disease countermeasures: preventivetreatments, diagnostics tests and therapeutic interventions. Molecularpathogenesis of most diseases remains elusive including the Spanish fluand AIDS. New molecular and bioinformatics tools are needed, inparticular systems biology-based approaches such as SRP measurementsusing the device of the invention.

11. SRP Tests for Cancer: Oral Tests for Cancer Screening and Lab Testsfor Cancer Diagnostics

Carcinomas of the skin, breast and prostate are among the most commonhuman malignancies. Current diagnostic methods typically involve aseries of tests. For example, a general screening test at POC is orderedby a family doctor during routine health testing (e.g., mammogram forBC). If this test is positive, the patient is referred to a CancerCenter. The Cancer Center surgeon performs a surgical biopsy that isexamined by a cancer pathologist using morphological criteria (e.g.,nuclear morphology, mitotic number) and molecular pathology (antibodystaining or DNA probing). If the biopsy is considered positive(malignant growth), the patient is referred for additional surgery. Thesurgical specimen is analyzed by a pathologist to assess the tumor gradebased on general grading scales (e.g., Gleason score for PC). Newresearch in personalized medicine aims to delineate the molecularmechanism of individual tumors using gene arrays or antibody staining.Following radio- and chemotherapy, diagnostic tests based on surgicalbiopsies and blood assays (not available for most cancers; PSA is usedfor PC) and are used to identify metastatic cancer. Many cancersurvivors suffer chronic pain and mental health problems for which thereare no objective tests. A related health problem is care-giver stress.

Tests for SRP are applicable across the cancer managementcontinuum—examples are listed below.

(1) Cancer Screening for Noninvasive Early Detection of Cancer.

-   -   Rapid oral test for BC and PC: a disposable LFIA. The test is        based on a multi-SRP assay optimized for a particular cancer (a        yes/no result). Some versions can discriminate metastatic        cancer.    -   Rapid semen test for PC: a disposable LFIA. The test is based on        a multi-SRP assay optimized for PC (a yes/no result). Some        versions can discriminate metastatic cancer.    -   Lab test for cervical cancer. The test is based on a multi-SRP        staining kit optimized for cervical cancer detection in a        standard cervical smear. Some versions can discriminate        metastatic cancer. The kit is used to stain a duplicate cervical        smear in parallel with the standard PAP test and provide a        yes/no result. The SRP test could be used as a decision support        for PAP (increased accuracy of cancer prediction) and ultimately        replace the PAP test. The accuracy of PAP would be improved in        two ways. First, positively staining cells with abnormal        morphology (i.e., PAP test reading made easier, faster, more        accurate). Second, positively staining pre-cancerous cells with        a normal morphology that cannot be detected by PAP. The        replacement could happen fast if the SRP test would provide an        outstanding benefit over PAP, e.g., discriminating metastatic        cancer.

(2) Cancer Diagnosis for Early Detection of Metastasis.

-   -   Lab test for a particular cancer. The test is based on a        multi-SRP staining kit optimized for the particular cancer and        corresponding tissue type. The kit is used to stain the biopsy        tissue and also the surgically removed tissue (tumor and        adjacent tissue). The test can discriminate metastatic cancer.    -   A staining kit for measuring individual SRP biomarkers. Results        (SRP profiles) interpreted using the computer software. Results        show a match with reference SRP profiles (i.e., discriminate        between not cancer, cancer, metastatic cancer). The results        indicate the molecular mechanisms of the particular cancer        (pathway profile). This information can be useful as a guide for        personalized therapy.

(3) Cancer Survivors for Improved Quality of Life.

-   -   Rapid oral test for cancer-related psycho-biological stress in        survivors.    -   Rapid oral test for cancer care giver stress.

12. Early PTSD Test

A large number of military service members suffer from PTSD andtraumatic brain injury (TBI). These diseases are complexpsycho-biological disorders that are hard to detect and quantify,particularly in early stages, before they can be assessed using standardpsychological/neurological tests. These standard tests require highlytrained medical personnel, therefore they are not practical for POC orat home monitoring. New tests for PTSD (under commercial development)include assays for saliva cortisol and saliva alpha-amylase (SAA). Theseanalytes are biomarkers for HPA and sympathetic nervous system pathways.SRP covers these pathways and many more pathways that are likely to beactivated by psycho-biological stress. Cortisol and SAA are also likelyto give false positive signals because they are increased by routinestressors.

13. Early Alzheimer's Test

Alzheimer's disease is the most common cause of dementia. The device ofthe invention can be used for rapid oral test for early Alzheimer's. Thetest is based on SRP biomarkers optimized for the disease.

14. Early Autism Test

Autism (ASD) is a rapidly growing health problem for kids in the US.Current goal for autism diagnostics is a test suitable for infants (<3yrs). Oral SRP test using the device of the invention would be moresuitable than tests currently under development.

15. Early Kidney Stones Test

A July 08 report predicted a sharp rise in kidney stones-related healthproblems in the US due to a higher incidence of dehydration caused byglobal warming. The device of the invention can be used for a rapid oraltest for early detection of kidney stones. The test is based on SRPbiomarkers optimized for kidney stones.

16. Early Kidney Disease Test

Chronic kidney diseases (CKD) are on a rise worldwide and earlydetection is essential for CKD management. The device of the inventioncan be used for a rapid oral test for early detection of CKD. The testis based on SRP biomarkers optimized for CKD.

17. Animal Wellness and Food Safety

There is a worldwide increase in emerging diseases and environmentalstressors related to factory farming, genetic modifications of crops andlife stock, antibiotics overuse, human impact on wildlife and globalwarming. This increase might be responsible for increased morbidity andmortality of domesticated and wild animals, wild species extinctions,and collapses of the US lobster fishery and the honeybee industry. Earlydetection of new diseases and environmental stressors is essential forpublic health, agricultural safety and wildlife protection andmanagement.

Factory farms keep animals in unhealthy conditions. Many animals arediseased and exposed to environmental stressors (crowding, injuries,antibiotics etc.). Organic farms are more likely to have less stressedanimals. Besides the ethical objections, products from stressed animalsare likely to have a lower nutrient value and might even present ahealth threat for consumers because stressed animals are likely to beill and the product might transmit diseases (e.g., mad cow disease), ormight contain metabolites that can cause health disorders because theyderegulate human cell function. For example, human metabolic disorderstriggered by animal hormones or immune disorders triggered by animalstress proteins. Currently, there is no objective test that regulatorsor consumers can use to assess whether a farm product is from a healthyor from a stressed animal. Such test would help consumers to chooseproducts from minimally stressed animals, and help regulators to searchfor unhealthy farm conditions.

A related issue is the safety of animal foods (pet food, farm feed). Ifanimal foods contain parts of diseased animals, it might transmit thedisease (e.g., BSE) or cause other types of health disorders.

The device of the invention can be used for SRP analysis of animal cellsand tissues related to disease, injury, environmental stress, and animalhealth and product safety, including:

1. Stress Tests for Animals and Animal Products (Milk, Egg).

-   -   The tests use the device of the invention modified for the use        with animal samples.    -   Saliva test for companion animals (dog, cat, horse); one smart        test device with species-specific cartridges or sample-specific        tests.    -   Milk test: a health test for dairy animals & milk quality test.        Could be made as an attachment for a milking machine or        equipment for milk quality test. SRP assay of milk cells and        fluids similar to the saliva SRP assay.    -   Egg test: a health test for poultry & egg quality test.    -   Male fertility test.    -   Urine test (a litter test for cats; farm animals).    -   Fish test. Fish biopsy or a surrogate small fish or invertebrate        living in the same habitat.    -   Shellfish test.    -   Beehive wellness test. The honeybee keeping industry has nearly        collapsed due to an unknown health threat (environmental factors        or disease).

2. Stress Test for Meat.

-   -   Cattle, pork, poultry, fish.

3. Stress Test for Animal Food.

-   -   Multi-SRP test that shows whether pet food and farm feed        products contain parts of stressed animals.

4. Stress Test for Sentinel Organisms.

Multi-SRP test of sentinel animals and wild animals found sick or deadof unknown causes. Increased stress indicates exposure to anemerging/occult disease or environmental stressors. For example,chickens are currently used as sentinels for infectious bird diseases.

18. Agricultural Safety

Plant diseases have increased worldwide possibly due to unsustainablefarming practices such as the widespread use of chemical fertilizers,pesticides and genetically modified plants, and global warming.Integrated monitoring of soil/plant/crop health could improve wellnessof farm crops, garden plants and house plants, and provide decisionsupport for the protection and management of wild plant ecosystems suchas forests and wetlands. Healthy crop plants are likely to produce cropsthat are more nutritious and case less health problems such as foodallergies. Currently, there is no objective test that regulators orconsumers can use to assess whether a crop (grains, fruits etc) is froma healthy or from a stressed plant. Such test would help consumers tochoose products from minimally stressed plants, and help regulators tosearch for unhealthy crop conditions.

Soil fertility correlates with the microbial richness of the soil, whichis naturally low in some soils (e.g., tropical soil) and becomesdepleted by agricultural use. Currently, there is a strong globalinterest in the restoration of soil fertility due to two factors: arapidly growing need for increased food production, and soil loss &depletion driven by global warming. However, there is no direct test formonitoring the wellness of soil microflora (the indirect test is theassessment of soil fertility based on crop yield and quality). There aretraditional soil probiotics such as compost or charcoal but no new,scientifically-based products that would rationally reduce stress insoil microbial systems.

There is an urgent need for a new technology that could assess andimprove soil fertility and crop health. The device of the invention toassess SRP technology meets these specifications.

19. Plant Disease and Food Research

Multi-SRP and SRP assays using the device of the invention can beadapted for soil and plant samples.

20. Stress Tests for Soil, Plants and Crops

Different soil samples can be analyzed using the device of the inventionwith the SRP biomarker panel optimized for soil microorganisms. AnotherSRP panel can be optimized for plants, focusing on stress markers forplant organelles (chloroplasts, mitochondria), commensal microorganisms(same as soil) plus stress markers that are in all species (e.g.,Hsp60/GroES or SOD). Candidate samples for plants include: roots,leaves, stems, sap. Samples for crops include: grains, fruits.

21. Environmental Safety Water Safety

Water safety affects public health, aquaculture, agriculture and naturalaquatic ecosystems. Standard methods for assessing water safety arebased on measuring several chemical and physical parameters (e.g., pH,temperature and turbidity), and levels of specific microbial andchemical contaminants. In England, a traditional method for water safetyis still used: the health condition of fish in the water (how many, howfast they swim past the observatory). Frogs are also traditionally usedas sentinel organisms for freshwater health. Since these methods do notprovide early warnings of declining water safety, new methods arecurrently being developed and tested. Several new methods measure thehealth condition of native aquatic microorganisms. One method uses aninfrared motility monitor. Another one, AquaSentinel, developed at theOak Ridge National Labs, uses a fluorescence reader to measure changesin algal bioluminescence.

The device of the invention can be used to provide SRP-based tests forwater safety, and provides several advantages such as: applicability toboth fresh water and seawater, broad-based sensitivity to changes inchemical, physical, biological water parameters including parametersthat are not measured by current sensors such as new types of airpollutants or agricultural run-off chemicals, early warning of incipientwater health deterioration (molecular stress responses precede changesin an algal fluorescent signature or motility), detection of emergingpathogens and biotoxins in aquaculture water that could be directlycorrelated to fish health, signature can be used to diagnose the natureof the water stress and to recommended countermeasures and used tomonitor the effectiveness of countermeasures in restoring water health.

Background—Air Safety

Microbial biosensors for airborne toxins have been introduced recently.Typically, these are genetically engineered bacteria or recombinantflies with a read-out gene (e.g., lux) linked to one of severalparticular toxin-sensitive genes (hsp70, DNA J). The disadvantage isthat recombinant organisms have to be manufactured for the biosensoroperation, and the small set of recombinant sensor genes in thebiosensor might not be sensitive to the large and diverse spectrum ofenvironmental stressors that affect people. The device of the inventioncan be used to provide SRP-based tests for air safety.

22. Space Technology

Key areas in space biology research that are useful with the device ofthe invention include: diagnostic and therapeutic technologies forastronaut health. The goal is to identify health risks of space flightand develop countermeasures to reduce those risks. The device of theinvention can also be used for fundamental space biology investigationsin microbial, plant and cell biology and animal physiology, i.e., howlife responds to gravity and space environments. Additionally, thedevice of the invention can be used to detecting life's signatures forfuture planetary missions to Mars, Europa and Titan.

23. Detection of Life's Signatures

Stress responses (SR) are universally present in all organisms on Earth.Responses to universal stressors are essential for life in general andcould be used in the search for life. The universal stressors arephysical-chemical gradients or agents commonly present in planetarygeological environments (e.g., electromagnetic radiation, radioactivity,temperature, gravity, heavy metals, water, CO₂). Universal stressors arealso generated by general biological processes such asself-organization, electron transfer and metabolism (e.g., heat, entropyand free radicals). Different biological systems might use verydifferent biochemical structures for SR but the different biochemicalstructures are likely to have the same or similar physical-chemicalfunctions. These general functions can be deduced through comparativestudy of biochemical structures that are commonly used for SR byterrestrial organisms. Components of the general physical-chemical SRfunctions are used as biomarkers for life.

For example, free oxygen and nitrogen radicals (RONS) are universalstressors produced by solar radiation as well as a by-product ofelectron transfer and metabolic reactions. RONS cause harm to all livingsystems through damage to macromolecular structures and shifts in redoxbalance. All terrestrial organisms have SR against RONS. Mostprokaryotes and eukaryotes use control RONS using superoxide dismutases(SOD). The physical-chemical function of SOD is carried out by the metalmoiety of SOD, which contains an antioxidant heavy metal (Fe, Mn, Cu,Zn). These and similar (Ni, Co) metals are candidate biomarkers forbiological anti-RONS responses.

Another universal stressor is water loss (desiccation). Most prokaryotesand eukaryotes have a SR for adapting to life without water(anhydrobiosis). If there was or is life on Mars, it had to deal withperiodical desiccation as well. The physical-chemical functionsunderlying the anhydrobiosis SR are candidate markers for lifedetection.

Bacterial and eukaryotic cells use organic osmolytics to cope withanhydrobiosis (glutamate, proline, glycerol, sucrose, trehalose,sorbitol, myo-inositol and glycine betaine). The physical-chemicalprinciple of the osmolytic compounds is structure-making (cosmotropic)function: they organize the water structure (hydrogen bonding) which isessential for structural integrity of biological membranes andbiopolymers (e.g., proteins). The known organic osmolytics listed above,and other cosmotropic compounds, are candidate biomarkers for biologicalresponse to osmotic stress and for anhydrobiosis.

24. Monitoring HIV/AIDS Risk and Treatment Outcome

HIV/AIDS is a priority public health condition⁶⁷. There are about 1.1million HIV infected people in the U.S. and 56,300 new HIV infectionsannually⁶⁷. Currently, two lab tests (CD4 count and viral load) are thegold standards for assessing AIDS risk and guiding cART⁶⁸⁻⁶⁹. CD4 counts350 (recently increased to 500) and 200 cells/mm³ are standardactionable thresholds for guiding cART⁶⁸. The CD4 test is expensive,invasive, time consuming, requires specialized equipment, highly trainedpersonnel and has to be repeated every 3-4 months. Thus, there is anunmet need for an affordable POC test for AIDS risk and guiding cART⁶⁹.

In one embodiment of the invention there is provided a rapid saliva testfor predicting AIDS risk treatment outcome that has been developed usingfollowing steps: (1) A candidate panel of 52 SR markers (see Tables 2and 3) was constructed using methods from Example 5. (2) The initialclinical validation of the panel used methods from Example 5 and salivasamples from HIV/AIDS patients (n=100) with CD4 counts >500, 200-500 and<200 cells/mm³. The AIDS risk test based on salivary SR biomarkers isradically different from current tests for the condition because itutilizes a new assay principle based on monitoring the physiologicalstatus of the patient. The assay principle was reduced to practice usingan immunoassay of SR biomarkers that monitor the physiological statusbased on cellular stress responses in saliva. The saliva test isnoninvasive, rapid and inexpensive. In contrast, current tests for AIDSprognostication monitor the immunologic, virologic or genetic status ofthe patient using blood samples and expensive laboratoryassays^(68-69,72).

The saliva test for AIDS prognostication has potential to providesignificant benefits for public health and substantial healthcaresavings in several ways: (1) Accelerating HIV/AIDS care and slowing thespread of HIV: Two rapid oral tests could be administered during routinehealthcare screening, an HIV diagnostics test followed by the salivatest in order to inform patients about their HIV status and AIDS risk ina single office visit so that they could receive care immediately.Pain-free, affordable oral testing is likely to increase the number ofHIV/AIDS patients connected to care and fewer people will be exposed toHIV: 54-70% of new HIV infections in USA are caused by people who arenot treated and engage in risky behaviors because they do not know thatthey are HIV infected⁶⁷. (2) Moving AIDS monitoring from lab topoint-of-care will greatly improve the delivery of clinical care andmedications in resource-limited settings where the standard tests forAIDS risk are not affordable and costly cART drugs are deliveredinefficiently, without lab tests^(69,74). (3) Enabling personalized HIVmedicine: frequent affordable testing of cART efficacy will facilitatedesigning and modifying cART for individual patients, which haspotential to improve treatment outcomes and decrease clinical costs⁷².

The following examples are intended to illustrate but not limit theinvention.

Example 1 Collection and Processing of Improved Saliva Samples forSaliva Diagnostics

This experiment provides an exemplary method for collecting andprocessing saliva samples that contain salivary cells. Although asdescribed below, assays based on the collection of human unstimulatedsaliva by spitting or brushing are exemplified, the assay principlescould easily be adapted to protocols that collect and process stimulatedsaliva samples, or use other devices and methods, or collect animalsaliva.

Sample collection. Unstimulated saliva samples were collected fromhealthy volunteers (5 women, 5 men, 8-53 yrs old). The subjects wereasked to brush teeth and have no food or beverage except water for 30minutes (min) before the collection. Two collection methods were used:(1) Spit was collected into a sterile container such as the 50 ml Falcontube. Subjects spit into the tube several times during about 15 minutes(min) until 3 to 6 ml of saliva was collected. During the collection,the tube was kept on ice. (2) A small oral brush described in FIG. 1 wasused to collect saliva by brushing teeth and gum surfaces on both sidesof the mouth for about 20 seconds. When a commercial oral brush (thePROXABRUSH Trav-ler, Sunstar Americas, Chicago, Ill.) was used for thiscollection method, the average collected saliva volume was 0.16±0.02 ml.To collect a larger volume of saliva, the brushing was repeated usingadditional brushes. A comparison of saliva samples collected usingself-brushing, and brushing performed by an assistant, showed that bothmethods yielded the same average sample volume and cellular composition.

Sample quality tests: (1) The adequate pH (6-8) was assessed by spotting5 μl of the spit on a pH test strip, or by pressing the tip of the gumbrush on a pH strip. Samples tested so far (n>100) had pH=7.2±0.6. (2)The adequate cell count (epithelial cells and leukocytes) was assessedusing one of the following methods. 1. Viable cell count. Saliva samplecollected by spitting was mixed thoroughly using a sterile 1 ml pipette.Using a sterile pipette tip, 10 μl from the middle of the tube wastransferred into a tube with 10 μl of 0.4% trypan blue in the phosphatesaline buffer pH 7.60 (PBS), mixed using the pipette tip, stained for 10minutes (min). 10 μl of the mixture was transferred into both chambersof a standard hemocytometer and viable cells (nucleated cells unstainedby trypan) were counted using ×100 magnification. Adequate viable cellcount was at least 0.2×10⁶ cells/ml. 2. Total cell count. Saliva samplecollected by spitting was mixed thoroughly using a sterile 1 ml pipette.Using a sterile pipette tip, 5 μl from the middle of the tube wassmeared across a small square (about 1 cm²) on a coated microscopy slide(a Silane Prep slide from Sigma, St. Louis, Mo.). Alternatively, salivacollected by the brush was smeared across about 1 cm length of theslide. Air dried saliva smears were fixed (1 dip in 2% formaline-1%acetic acid-80% ethanol followed by 10 dips in water) and stained usinghematoxylin and eosin (H&E stain, Sigma): 10 dips in hematoxylinfollowed by 10 min in water, 10 dips in 80% ethanol, 10 dips in eosin,10 dips in 95% ethanol, air dry, 2 dips in xylene, coverslip. Cellsstained with H&E were counted at ×100 magnification. The adequate cellcount was at least 2,500 cells per smear (>5×10⁵ cells/ml). Normalsaliva smears typically contained nucleated epithelial cells andleukocytes as well as about 10% of epithelial cells without nuclei. (3)The adequate cellular composition of the sample was determined based onmicroscopic inspection of the H&E saliva smear. A typical saliva samplecollected by spitting had about 60% epithelial cells and 40% leukocytes(monocytes, lymphocytes and granulocytes). Brush-collected samplestypically had about 50% epithelial cells and 50% leukocytes. Inaddition, all normal saliva samples also contained variable amounts ofresident bacterial and fungal cells (about 10⁵-10⁷ microbial cells/ml).The salivary microbial cells were associated with the mammalian cells,or formed microbial clumps or were dispersed as single cells.

Salivary cell smears on microscopy slides (“smears”). Smears are usefulfor salivary diagnostics in multiple ways. They enable H&E analysis andthe quantitative measurements of cell-associated molecular biomarkers(proteins, peptides, mRNA, DNA, small molecules such as eicosanoids, orreporters) in salivary epithelial cells, leukocytes and microbial cells.Proteomic markers and small molecules can be measured using theimmunocytochemical staining (“the ICC assay”) or by other assays such asreporter assays or in situ nucleic acid hybridization. Smears wereprepared using following methods. 1. Saliva collected by spitting. Thesample was thoroughly mixed in a tube using a 1 ml sterile pipette.Using a sterile pipette tip, 40 μl from the middle of the tube wastransferred on a coated slide and the spot was immediately spread acrossthe whole slide using the tip. The tip was tilted at a sharp angle tofacilitate the spreading. In some experiments, smaller volumes of salivawere spread in separate areas of the slide to compare different salivasamples on the same slide (e.g., four smears, 10 μl each). 2. Samplescollected using oral brush. Immediately after removing from the mouth,the brush was smeared across the length of a coated slide. The samebrush was used to prepare additional slides (typically, 4 slides perbrush). Additional brushes were used to collect more saliva and preparea full set of slides for biomarker analysis (e.g., 25 slides wereprepared using 7 brushes). Smears prepared sing the spit or brushmethods were air dried at room temperature (RT) for at least 30 min,fixed in 10% normal buffered formalin for 10 min, followed by 3×5 minrinses in PBS, 5 min in water and 5 min each in 80%, 95% and absoluteethanol. Dry fixed slides were stored in a standard histology slide boxat RT. The fixed slides were stable for over 3 years based on the ICCassay using control antibodies.

Salivary ICC Assay. The main advantages of ICC are sensitivity (specificstaining of single cells corresponding to 0.1-1 pg/ml antigenconcentration) and specificity (each marker stains specific cell typeand has a characteristic cellular localization). To demonstratecompatibility with ICC, representative saliva smears were stained withcontrol antibodies. Positive control antibodies were specific forantigens consistently expressed by salivary epithelial cells orleukocytes. As positive controls, EMA (a membrane antigen on about 30%salivary epithelial cells; mouse IgG2a, 0.2 μg/ml, Biogenex, San Ramon,Calif.) and CD68 (a cytoplasmic antigen in salivary monocytes, B cellsand neutrophils, mouse IgG1, 0.5 μg/ml, Dako, Carpenteria, Calif.) wereused. Negative control antibodies were mouse monoclonal antibodies (Mab)and rabbit polyclonal antibodies (Pab) specific for irrelevant antigensthat are not present in salivary cells. Negative controls matched theconcentration, species and type of positive control antibodies andanti-biomarker antibodies. As negative controls, a mouse IgG1 Mab(anti-digoxigenin, 0.5 μg/ml, Santa Cruz Biotechnology, SCBT, SantaCruz, Calif.) and a rabbit Pab (anti-Drosophila armadillo, 0.2 m/ml,SCBT) was used. A new protocol was developed to enable ICC assay ofsaliva smears. Before staining, dry slides were scored with a diamondpen to outline sections for the application of different antibodies(typically 4 control antibodies were applied to one slide; in antibodytitration experiments, 8 antibodies were applied to one slide). Assaysteps: (1) Based on extensive testing, it was determined that commonlyused antigen unmasking methods that use heat treatment (citrate orglycin buffers, 95-100° C. for 10-20 min) were not suitable for salivasmears because >80% cells fell off slides during the heat treatment.Therefore, a new method was developed for antigen unmasking in salivasmears: slides were placed in 20 mM citrate, 0.1 mM EDTA buffer (pH 3.0)at 37° C. for 60 min, followed by 5 min rinses in water and PBS pH 7.60.(2) Slides were blocked using PBS with 7% normal goat serum for 30 min.(3) Based on extensive testing (64 rabbit and goat Pabs, 25 mouse andrat Mabs), we determined that (i) Mabs (whole culture supernatants,ascites or purified immunoglobulins) and immunoaffinity-purified Pabswere suitable for salivary immunoassays whereas (ii) Pabs in the form ofthe whole serum (nonimmune serum or antiserum) or the immunoglobulinfraction of a whole serum, were unsuitable for salivary immunoassaysbecause they contained antibodies that strongly stained 10-30% salivarymicrobial cells even when highly diluted (<10:1000). The anti-microbialaffinity of whole serum has never been reported previously, probablybecause typical samples for immunoassays are sterile tissue culturecells, blood cells and fixed tissues that were stripped of residentmicrobes. Based on the results, only Mabs or affinity-purified Pabs wereused for all saliva immunoassays (ICC or other assay formats). (3)Optimal concentrations of primary antibodies (the EMA, CD68,digoxigenin, armadillo antibodies) were diluted in PBS, pH 7.60 with 1%bovine serum albumin (BSA) and applied to individual sections on blockedslides after draining off the blocking solution and dividing the cellsmear into fields by wiping between the outlined sections using asharply folded paper tissue. The total antibody volume was 0.3 ml perslide. Afterwards, slides were placed in a humidified chamber at 4° C.for 16-20 hrs. (4) After 3×5 min rinse with PBS, a secondary antibodywas applied for 90 minutes at RT (a biotinylated goat antibody againstmouse and rabbit IgG, Biogenex, 1:20 in PBS-BSA), followed by 3×5 minrinse with PBS, enzymatic conjugate for 30 min at RT (astreptavidine-alkaline phosphatase conjugate, Biogenex, 1:20 inPBS-BSA), 3×5 min rinse with TBS (50 mM Tris, 150 mM NaCl, pH 7.60),chromogen (Fuchsin, Dako), 5 min water rinse, hematoxylin stain for 1min, 15 min water rinse and two 5 min rinses with 95% ethanol. Air driedslides were rinsed in xylene and cover-slipped before a microscopicexamination at ×100 magnification. (5) The staining intensity wasquantified using computerized image analysis. 3 representative imageswere captured in each stained section and areas with at least 100epithelial cells or leukocytes were outlined in each image. The meanoptical density (MOD) in the outlined area, and the percent of thestained area (PA), were determined by applying a color file to theimage. The same color file was applied to all images to ensureconsistent MOD and PA measurements. The staining intensity (SI) wascalculated as SI=MOD×PA. The mean SI was calculated for 3 images perstain. To determine the reproducibility of the assays, the mean SI wasmeasured in 12 duplicate stains produced in the same and consecutiveassay runs. The measurements were compared using linear regressionanalysis to calculate 95% confidence interval for the mean ofdifferences. The coefficient of variation (CV) was 9.8%, demonstratingthat the measurement was reproducible. Results of the control stainingshowed critical parameters for the ICC assay: sensitivity 0.1-1 pg/ml(based on the EMA antigen concentration), specificity (no staining withnegative control antibodies), intra- and inter-assay reproducibility(<10% CV for the mean SI measurements).

Saliva protein lysates. The lysates enable detecting protein, peptideand small molecule biomarkers present is cell-free saliva and releasedfrom solubilized salivary cells. Lysates can be analyzed using ELISA,protein blots, mass spectrophotometry, chromatography or other types ofassays. As explained in the ICC assay protocol above, antibodiessuitable for saliva immunoassays are Mabs or immunoaffinity-purifiedPabs. Lysates were prepared using two methods. (1) Spit-based samples: 1ml of a 2× concentrated lysis buffer (LB) was added per 1 ml saliva; thesample was thoroughly mixed using a sterile 1 ml pipette and kept on icefor 30 min. The final concentrations in the lysate were 1 mM EDTA, 1 mMPMSF, 1 mg/ml N-ethylmaleimide, 0.02 mg/ml ovatrypsin inhibitor, 0.1mg/ml aprotinin, 6 mg/ml 4-aminobenzamidine dichloride and a cocktail ofmammalian phosphatase inhibitors from Sigma diluted in PBS. (2) Salivawas collected using the oral brush (FIG. 1) as described above.Immediately after the collection, the brush was removed from the handleand suspended in 2×LB. Four brushes (the PROXABRUSH Trav-ler, SunstarAmericas) were mixed with 0.3 ml of the LB in one microcentrifuge tubeby swirling the brushes in the buffer for 30 seconds. The brush was keptin the buffer on ice for 30 min, and then removed. After lysis,insoluble material was removed by centrifugation at 12,000 rpm and thesupernatant was transferred to a new tube and immediately frozen at −80°C.

Saliva ELISA. The ELISA assay complements the ICC assay of biomarkers insalivary cells by measuring soluble biomarkers. The main benefits ofELISA are sensitivity to 1 pg/ml biomarker concentrations, consistenthigh throughput and reliable metrics (pg/ml concentration) that clearlyshow the success of clinical diagnostic studies. To demonstratecompatibility with ELISA, we used a multiplexed MultiBead ELISA(Inflammatory Panel, Assay Designs, Ann Arbor, Mich.) to measure 8control proteins (IL-1beta, IL-4, IL-6, IL-8, IFN-gamma, TNF-alpha) andsmall molecules (eicosanoids PGE2 and TXB2) with known concentrations innormal saliva⁸⁻¹⁰. Calibration curves for the analytes were constructedusing serial dilutions of purified standards first in buffer (PBS-BSA)and then saliva matrix (the saliva protein lysate described above, apool from several subjects). The assay was optimized to reach benchmarkvalues of critical assay parameters: limit of detection at 1-10 pg/ml,linear range 10 pg-10 ng/ml, recovery (assay interferents), specificity(no signal with irrelevant purified antigens), intra- and inter-assayreproducibility (<10% CV for the mean measurements of duplicate samplesin the same and in consecutive assay runs). The optimized assay was usedto measure the 8 control analytes in normal saliva lysates fromindividual subjects.

Saliva DNA lysates. These lysates enable measuring DNA released fromsolubilized salivary cells. DNA prepared from the lysates can beanalyzed using PCR, DNA blots or other types of DNA assays. Assays ofsaliva DNA have potentially wide applications in human and animaldiagnostics including pharmacogenomics (individualized testing of drugsafety and efficacy), testing for genetic disorders (diseaseprognostics), paternity and forensics. Although as described below, aprotocol for DNA preparation is exemplified, the protocol principlescould easily be adapted to protocols that prepare RNA. Saliva DNAlysates were prepared from saliva collected by spitting in a tube or byoral brushing as described above. The objective was to develop a simplemethod that could be used in field conditions using reagents and lysatesthat are stable at RT, and can be later processed in a laboratory toprepare DNA suitable for PCR amplification. Six such methods weredeveloped: (1) 0.1 ml of a 5× concentrated lysis buffer (LB) was addedto 0.5 ml spit in a sterile tube, the sample was thoroughly mixed usinga sterile 1 ml pipette or vigorous shaking Final concentrations in thelysate were 10 mM Tris-HCl, 10 mM EDTA, 0.1% sodium dodecyl sulphate(SDS). Fresh concentrated Proteinase K (PK, Qiagen, Valencia, Calif.)was added to 10 μg/ml final concentration. The lysate was incubated at50° C. for 1 hr, boiled for 3 min, 25 μl of 5M NaCl was added to 0.2 Mfinal concentration, the sample was mixed with 1 ml absolute ethanol,incubated at RT for 20 min, centrifuged at 14,000 rpm for 10 min, thepellet was rinsed with 70% ethanol, air dried and dissolved in 50 μl of10 mM Tris-1 mM EDTA buffer pH 7.60 (TE). (2) Same as Method 1 but theLB contained diluted PK and was stored at RT for 2 days before use. (3)Same as Methods 1 or 2 but after boiling, 50 μl of 5M iced potassiumacetate (pH 4.8) was added, the sample was mixed thoroughly, iced for 30min, centrifuged at 14,000 rpm for 15 min, the supernatant wastransferred to a new tube, mixed with 1 ml of absolute ethanol,incubated at RT for 20 min, centrifuged at 14,000 rpm for 10 min, thepellet was rinsed with 70% ethanol, air dried and dissolved in 50 μl ofTE. (4) Same as Methods 1-3 but the lysate was incubated at RT for 18hrs instead of at 50° C. for 1 hr. (5) 0.5 ml of spit was mixedthoroughly with 0.1 ml of a concentrated lysis buffer by vortexing orvigorous shaking Final concentrations in the lysate were: 50 mM NaOH, 10mM EDTA and 0.025% SDS, and the pH was about 12. The lysate was storedat RT for 8 days without additional mixing. On day 9, the lysate wasboiled for 10 min, iced to RT, neutralized to pH 7.8 by adding 5 μl of2M Tris-HCl pH 7.0 and 25 μl of 1M HCl. The neutralized lysate contained50 mM NaCl. Insoluble material was pelleted by centrifugation at 14,000rpm for 5 min, the supernatant was transferred to a new tube, 15 μl of5M NaCl was added to final concentration of 0.2 M, the lysate was mixedwith 1 ml of absolute ethanol, incubated at RT for 20 min, centrifugedat 14,000 rpm for 10 min, the pellet was rinsed with 70% ethanol, airdried and dissolved in 50 μl of TE. (6) Same as Methods 1-5 but salivawas collected using an oral brush (FIG. 1) as described above.Immediately after the collection, the brush was removed from the handleand suspended in LB. Four brushes (PROXABRUSH Trav-ler, SunstarAmericas) were mixed with 0.1 ml of 5×LB in one micro centrifuge tube byvortexing or by 10× swirling the brushes in the buffer. To estimate theDNA concentration and the molecular weight (MW), 5 μl each of a DNAstandard (HyperLadder I, Bioline, Taunton, Mass.) and the saliva DNAwere analyzed using a standards 0.7% agarose TBE gel with 0.5 m/mlethidium bromide. The average yield per 1 ml saliva was: 3±1 μg of highMW DNA (>20 kbp) for Methods using PK (1-4) and 1.5±0.5 μg ofmediate-low MW DNA (1-20 kbp) for Methods using NaOH (5). To showcompatibility with PCR, a 500 bp fragment of the human IFN-beta gene wasamplified in the different saliva DNA preparations using followingprimers: 5′ ATG ACC AAC AAG TGT CTC CTC CAA A and 5′ GTT TCG GAG GTA ACCTGT AAG TCT G, and standard hot-start reaction conditions using 1.5 mMMgCl₂, 40 Cycles: 94° C. (45 sec); 60° C. (60 sec); 72° C. (60 sec),then final extension at 72° C. (10 min). The PCR product and a DNAstandard were visualized using a standard 2% agarose gel stained withethidium bromide.

Salivary cells. Live, fixed or permeabilized salivary cells are usefulfor salivary diagnostics by enabling the detection of molecularbiomarkers using flow cytometry (FCM) or immunofluorescence assays. Asexplained in the ICC assay protocol above, antibodies suitable forsaliva immunoassays are Mabs or immunoaffinity-purified Pabs. Salivarycells were prepared using the following procedure: Spit was diluted 1:1with a staining buffer (SB: phosphate buffered saline, pH 7.6, 2% BSA,0.1% azide) and centrifuged at 300 g for 5 min. Brush-collected salivarycells were released into SB (10-30 brushes submerged in 1 ml SB, 5 minon ice on a rocker), and centrifuged as above. The cell pellet wassuspended in a minimal volume of SB (e.g., 0.1 ml), 5 μl were removed toperform a cell count, and to determine >90% cell viability using trypanblue exclusion as described above. The average yield was about 6×10⁵mammalian cells/ml spit, and about 3×10⁴ cells/brush. To demonstratecompatibility with FCM analysis, duplicate samples of salivary cellswere stained with control antibodies using a standard protocol forstaining of live cells: The cell suspension was diluted to get a finalcell concentration of about 10⁶ cells/ml, incubated with anti-Fcreceptor antibody (CD32, SCBT, 1 mg/ml, 10 min), divided into stainingsamples in microcentrifuge tubes (at least 1×10⁵ cells/sample),centrifuged at 300 g for 5 min at 4° C., resuspended in 0.1 ml with aFITC-labeled primary antibody diluted in SB (1:5 diluted CD68-FITC andnormal mouse IgG₁-FITC, SCBT), mixed and incubated on ice in dark for 30min, rinsed 3× with SB and transferred into a Falcon 2052 tube with 0.4ml SB before FACS analysis.

Example 2 Production of Reference Reagents and Materials for SalivaryDiagnostics

This experiment provides an exemplary method for the production of novelreference reagents and materials for saliva diagnostics by inducingcellular stress in cultured normal salivary cells by in vitro treatment.

Preparation of stressed salivary cells. Saliva samples (6 ml) weresimultaneously collected from 3 healthy volunteers (1 man and 2 women,19-52 years old) using the spit method from Example 1. The acceptabilityof the samples was immediately evaluated using a pH test and H&E stainas described in Example 1. The samples were combined into a “Normal (N)pool”. A portion the N pool was processed into smears and or lysatesusing protocols from Example 1 (e.g., 3 ml was processed into 75smears). The remaining N pool (15 ml) was divided into 5 cultures: (3 mlsaliva, ˜1×10⁶ viable cells/culture). The cultures were maintained insterile Petri dishes (polystyrene, 60 mm×15 mm, Sigma) in a standardcell culture incubator at 37° C. for 18 hrs without adding culturemedium. The cultures contained whole saliva with all normal salivarycell types: epithelial cells, monocytes, B and T lymphocytes,granulocytes, fungi and bacteria. Each culture was treated by adifferent environmental stressor: (1) Hypersalinity was induced byadding 150 mM NaCl and incubation for 18 hrs, as previously used forcultured kidney cells¹¹. (2) Oxidative stress was induced by adding0.01% azide and 0.2 M ethanol and incubation for 18 hrs. (3) Heat shockwas induced by incubation at 44° C. for 2 hrs followed by incubation at37° C. for 16 hrs. Similar conditions were previously used to heat shockHeLa cells¹². (4) Cold shock was induced by freezing saliva at −80° C.for 2 hrs (3 sterile cryotubes, 1 ml saliva/tube), thawing on ice byadding 1 volume of warm growth medium (RPMI with 20% fetal calf serum),transfer into a sterile Petri dish and incubation at 37° C. for 18 hrs.(5) Desiccation was induced by reducing the culture volume to 1 ml usingprogressive evaporation during 2 hrs, followed by 16 hr incubation atthe same volume. A portion of each treated culture (1 ml from treatments1-3, 2 ml from treatment 4 and 0.3 ml from treatment 5) was processedindividually as “Treated (T1-T5)”. Remaining treated cultures werecombined into a “Stressed (S) pool” (about 10 ml) before processing intosmears on slides. The smears were produced using methods from Example 1.

To determine if the treatments induced cellular stress, 40 SR markerswere measured in smears of N pool, S pool and T1-T5 using the ICC assayprotocol described in Example 1. The primary antibodies were a pool ofantibodies against 40 SRP markers (see Table 2) and control antibodieswere as described in Example 1. The treatment was considered successfulif the average SR marker level was over 3 fold higher in treated saliva(T1-T5, S pool) than in the N pool, see FIG. 2.

Although stressed salivary cells can be prepared from using one donorand a single environmental stressor, the preferred method describedabove is based on the combination of saliva samples from several donorstreated using 2 or more different environmental stressors. The preferredmethod produces a broad-based cellular stress in saliva, as salivarycells from different genetic backgrounds respond to the variousstressors by activating multiple stress response pathways. Thebroad-based cellular stress results in altered levels of numerousbiomarkers that are affected by cellular stress. The induced biomarkersare present both within salivary cells and also secreted into theculture medium. FIG. 3 documents that treatment of saliva cells by thepreferred method increased levels of SR biomarkers more than 20-foldindicating a broad-based cellular stress. FIG. 4 shows that at least 50individual SR markers were induced by the preferred method indicatingbroad-based activation of the 10 principal SR pathways monitored by the40 markers (see Table 1).

FIG. 5 shows that (i) the SR marker profile induced by desiccation wasreproducible in salivary cells from different subjects, (ii) SR profilesdiscriminated between effects of desiccation, heat shock andhypersalinity and (iii) three SR markers were sufficient to discriminatebetween effects of desiccation and heat shock.

Broad-based cellular stress in saliva produced by the preferred methodis directly relevant to clinical salivary diagnostics because a verysimilar broad-based cellular stress was found in saliva samplescollected from subjects with disease or trauma, see FIGS. 3 a-d.

The 40 SR markers were detected in volunteers with inflammatoryconditions that commonly affect the oral cavity (gingivitis andperiodontitis, n=2). These volunteers typically had about 5-10% higherconcentration of salivary leukocytes. The average SR markerconcentration was less than 1.1-fold higher in the saliva withinflammatory conditions than in saliva from subjects without thecondition (n=10), which is a statistically insignificant. This resultindicates that salivary diagnostics of disease or trauma is not affectedby common oral inflammatory conditions.

Example 3 Development of Assays for Salivary Biomarkers

This experiment provides an exemplary method for the development of asalivary biomarker assay. The method uses reference reagents andmaterials prepared as described in Example 2. Two types of laboratorysaliva immunoassays are exemplified, the immunocytochemical (ICC) assayand the ELISA assay.

The ICC assay. Methods in Example 2 were used to prepare referenceslides for the assay: salivary cell smears of the N and S pools. Thereference slides were first used to determine the optimal concentrationof the tested anti-marker antibody. The reference slides were preparedusing 20 μl of the N pool horizontally smeared across the top of theslide, and 20 μl of the S pool smeared across the bottom of the slide.Before staining, the slide was vertically divided into 4 sections byscoring the opposite side of the slide with a glass pen so that eachsection contained the N pool on the top and the S pool on the bottom.The sections were stained with 4 serial dilutions of the anti-markerantibody using the ICC staining protocol from Example 1. Parallel slideswere stained with the control antibodies described in Example 1. Theoptimal concentration of the anti-marker antibody was identified basedon the smallest detectable specific staining in the N pool, and thehighest signal ratio between N and S pools. The sensitivity of the assaywas shown based on the detection of single stained cells. Thespecificity of the assay was shown as the absence of staining with thenegative control antibody. The reproducibility of the assay was shown as<10% CV for repeated measurements of the mean staining intensity induplicate samples N and S pools in the same assay run and in 3consecutive runs. Optimal concentrations of 52 SR markers determinedusing this method are in Table 5.

TABLE 5 Antibodies for the Detection of SR Biomarkers in Salivary CellsANTIGEN ANTIBODY M DF ASK-1 AAP-480 1 1000 Endorphin beta MAB0905 1 100CARD 9 905-188 1 600 Caspase 8 AAP-118 1 50 Cyclin D1 KAM-CC200 1 100Cox-2 sc-7951 2 600 Cytochrome 450 MFO-100 1 600 CYP450 reductaseOSA-300 1 1000 EGFR sc-03 2 150 Ferritin A0133 3 3000 Fos 905-640 1 10Glucocorticoid receptor sc-8992 2 600 GroEL SPS-875 1 600 Grp58 SPA-5801 1000 Grp75 SPA-825 1 50 GSTp A3600 3 1000 HO-1 SPA-895 1 4000 HSF-1SPA-901 1 600 Hsp 25 SPA-801 1 400 Hsp 27 SPA-800 1 100 Hsp 40 SPA-400 1150 Hsp 60 SPA-804 1 750 Hsp 70 SPA-810 1 1000 Hsp 90 SPA-830 1 20 IL-1beta sc-7884 2 800 IL-6 sc-7920 2 1000 IL-8 sc-7922 2 800 IL-10 sc-78882 800 iNOS KAP-NO001 1 30 Jun KAP-TF105 1 150 Laminin PU078-UP 1 150Leptin receptor sc-8391 2 10 Metallothionein MO639 3 15 Mekk-1KAP-SA001E 1 100 Mek-1 KAP-MA010E 1 400 MMP-9 905-486 1 1500 p53KAM-CC002 1 50 PBR sc-20120 2 400 Saliva alpha amylase sc-25562 2 500Serotonin sc-73024 2 10 Serotonin R1A 905-741-100 1 100 Substance Psc-58591 2 100 SOD Cu SOD-100 1 800 SOD EC SOD-106 1 400 SOD Mn SOD-1101 600 TGF beta sc-7892 2 400 VIP sc-20727 2 100 ALR sc-33219 2 250 AQP5sc-28628 2 600 BGT-1 B1082-10 4 100 SAPK KAP-SA011 1 50 p38-MAPKKAP-MA022 1 30 M, manufacturer: 1-Assay Designs, Ann Arbor, MI. 2-SantaCruz Biotechnology, Santa Cruz, CA. 3-Dako, Carpinteria, CA. 4-USBiological, Swampscott, MA. DF, dilution factor.

The ELISA assay. Methods in Examples 1 and 2 were used to preparereference samples for the assay: protein lysates of the N and S pools.The tested biomarker was analyzed using a commercial ELISA assay usingmethods and the control ELISA assay as described in Example 1. The assaywas optimized to achieve benchmark values for critical assay parametersas described in Example 1. For large marker panels, the development of amultiplexed ELISA assay (e.g., the 8-plex MultiBead ELISA, AssayDesigns) was preferred over the single ELISA assay based on nearly20-fold lower sample volume per analyte and lower cost per sample forthe multiplexed ELISA.

Example 4 Determination of Baseline Concentrations for SalivaryBiomarkers

This experiment provides an exemplary method for measuring baselineconcentrations of saliva biomarkers using two complementary assays, ICCfor cell-associated biomarkers in saliva smears, and ELISA for solublebiomarkers in saliva lysates. The method uses reference reagents andmaterials prepared in vitro as described in Example 2.

Saliva samples (3 ml) were collected from 10 healthy volunteers at 6time points (3 days, 8 am and 3 pm) and used to prepare “individualsmears” and “individual lysates” using methods from Example 1. On thefirst collection day, 1 ml aliquots of each sample were combined (20 ml)and used to prepare smears and protein lysates from N and S pools usingMethods from Examples 1 and 2. Methods from Example 3 were used tovalidate saliva ICC and ELISA assays of the tested biomarker.

Using the validated ICC assay, the tested biomarker was measured intriplicate slides of the individual smears. Smears of the N pool and Spool were used as reference slides with normal and increased levels ofsaliva biomarkers. The mean SI was determined for each smear using imageanalysis method from Example 1. The baseline was calculated as theaverage of the mean SI measurements in the individual samples.Individual and daily variability was determined as the standarddeviation from the baseline.

Using the validated ELISA assay, the tested biomarker was measured induplicate samples of individual lysates. Protein lysates of the N pooland S pool were and used as reference samples with normal and increasedlevels of saliva biomarkers. The baseline was calculated as the averageconcentration in the individual samples. Individual and dailyvariability was determined as the standard deviation from the baseline.

Example 5 Construction of a Biomarker Panel for Salivary Diagnostics

This experiment provides an exemplary method for constructing abiomarker panel that is useful for salivary diagnostics of healthdisorders.

Potential markers. Potential markers were identified using two methods:(1) Articles describing the molecular mechanism of cellular stressresponses (SR) associated with health disorders were collected frompeer-reviewed scientific literature. Meta-analysis of the articles wasused to select potential biomarkers based on their association with oneor more universal SR pathways that are activated in different cell typesduring more than one health disorder⁴⁻⁷. Ten universal SR pathways aredescribed in Table 1. (2) Protein lysates of the N pool and S pool wereprepared using methods from Example 2. The lysates were analyzed toidentify differentially expressed proteins and peptides using a methodwith a sufficiently high sensitivity and peptide separation to enablereliable sequencing and identification of peptides in a complex proteinmixture such as the saliva lysate, for example the isotopic labelingcoupled with liquid chromatography tandem mass spectrometry(IL-LC-MS/MS). Potential biomarkers were identified based on more than2-fold difference in the concentration between the S and N pools.

Candidate marker panel. Reference slides and protein lysates wereprepared from the N pool, S pool and treated cultures T1-T5 usingmethods from Examples 1 and 2. Methods from Example 3 were used tovalidate ICC and ELISA immunoassays for potential saliva markers.Methods from Example 4 were used to measure the normal baseline andvariability of the potential markers. Candidate biomarkers were selectedfrom the potential biomarkers using following criteria: (1) Each markerhad a stable baseline in normal saliva based on less than 2-foldindividual and daily differences in the marker concentration. In suchmarkers, the ratio between the baseline concentration and the standarddeviation of the baseline is less than 0.65. (2) The concentration ofeach marker was more than 3-fold different between the S and N pools.Preferred markers had more than 3-fold increased concentration in the Spool relative to the N pool. (3) When combined into a panel, the markersdiscriminated between the T1-T5 saliva samples. A panel of 52 candidatesalivary biomarkers identified using this method is shown in Table 5 andFIGS. 2-5.

Initial clinical validation. A small-scale clinical study was used todemonstrate that a candidate marker panel had a potential diagnosticvalue for a specific medical condition. Clinical saliva samples and goldstandard indicators of the medical condition were collected usingmethods from Example 1. A practical limit for the volume of clinicalsaliva samples was about 3 ml since in many medical conditions patientscannot produce as much saliva as healthy people. The saliva samples wereprocessed into saliva smears and protein lysates using methods fromExample 1. Individual biomarkers were measured in the smears and lysatesusing the validated ICC and ELISA assays. The assays used referenceslides and lysates with normal and increased levels of saliva biomarkers(the N pool and S pool) prepared from normal saliva using methods fromExamples 1 and 2. The discrimination of the medical condition using thesaliva biomarker panel was determined using correlation analysis withthe gold standard indicator. The diagnostic accuracy of the salivabiomarker for the threshold value of the gold standard indicator wasdetermined using the Receiver Operator Characteristics (ROC) curveanalysis, which provided the criterion values (cutoff sensitivity andspecificity values that divide true negatives and true positives) andthe Area-Under-Curve value (AUC)¹³⁻¹⁵. Optimized biomarker panel wasconstructed by combining a minimal number of markers that classified themedical condition with the greatest AUC value and the most narrow rangeof criterion values.

Large-scale clinical validation. To efficiently measure biomarkers inlarge sample sets, a multiplexed ELISA assay for the optimized salivabiomarker panel was produced using a commercial assay platform such insuch as MultiBead ELISA (Assay Designs) and methods from Example 3. Theassay used reference lysates with normal and increased levels of salivabiomarkers (the N pool and S pool) prepared from normal saliva usingmethods from Examples 1 and 2. Biomarker measurements obtained by themultiplexed ELISA were used to construct the final biomarker panel thataccurately discriminated the specific medical condition and was notaffected by potentially confounding variables such as gender, age andother medical conditions.

The final biomarker panel was used for the forward design of acommercial diagnostic test using a mature assay technology with provenacceptability by regulators and customers such as the lateral-flowimmunoassay (LFIA)¹⁶⁻¹⁷. The multiplexed ELISA assay was used as thereference assay in the testing of the commercial test. The prototypetest was optimized using reference lysates with normal and increasedlevels of saliva biomarkers (the N pool and S pool) prepared from normalsaliva using methods from Examples 1 and 2.

Example 6 Saliva Test for Monitoring Hydration Status

A rapid saliva test for >3% dehydration has been developed usingfollowing steps: (1) A candidate panel of 52 SR markers (see Table 5)was constructed using methods from Example 5. (2) The initial clinicalvalidation of the panel used methods from Example 5 and a laboratorystudy of dehydration induced in healthy volunteers (n=15) by exercise inheat without fluid intake. The study design discriminated betweeneffects of dehydration and exercise-heat. Stable euhydration before thetrial was documented based on consistent body mass (±1%), plasmaosmolality <290 mOsmol/kg, urine specific gravity <1.02 for 3 days⁵⁶.During the trial, progressive dehydration from 1 to 6% was monitored by1-4% weight loss. Samples of saliva, blood and urine were collected at 9time points. The blood and urine samples were used for standardlaboratory tests of the hydration status including the gold standardtest (plasma osmolality). The clinical saliva samples were collected andprocessed into smears and protein lysates using methods from Example 1.Individual 52 SR markers were measured in the smears and lysates usingoptimized ICC and ELISA protocols as described in Example 5. The assaysused reference slides and lysates with normal and increased levels ofsaliva biomarkers (the N pool and S pool) prepared from normal salivausing methods from Examples 1 and 2. The SR marker measurements werecorrelated with plasma osmolality to determine whether the SR markermeasurements were significantly related to plasma osmolality and notaffected by potentially confounding effects of exercise-heat, gender andsampling variables. The diagnostic accuracy of the SR markers for theplasma osmolality threshold (296 mOsmol/kg indicating 3% dehydration)was determined using the ROC curve analysis as described in Example 5. Aminimal panel of SR markers that had the best diagnostic accuracy for≧3% dehydration was selected using methods from Example 5.

(3) Methods from Example 5 and a large clinical study of dehydration(n=100) were used to construct the optimized SR marker panel. (4) Theoptimized SR marker panel was used to produce a prototype LFIA device.The prototype was optimized using reference saliva lysates as describedin Example 5. The optimized prototype was tested extensively usingclinical saliva samples to demonstrate reliability, accuracy,applicability for field use and regulatory requirements. The multiplexedELISA assay was used as a reference assay for the LFIA. The LFIA deviceshowed actionable levels of dehydration based on plasma osmolalitythresholds: normal (plasma osmolality <290 mOsmol/kg), moderatelydehydrated (2-3% dehydration, osmolality 291-296) and severelydehydrated (dehydration >3%, osmolality >296), see FIG. 6. The benefitof identifying moderate dehydration is that it can be treated in thefield by simple oral rehydration that prevents progression to severedehydration that might require hospitalization and intravenousrehydration.

Example 7 Saliva Test for Monitoring HIV/Aids Risk and Treatment Outcome

A rapid saliva test for predicting AIDS risk treatment outcome has beendeveloped using following steps: (1) A candidate panel of 52 SR markers(Table 5) was constructed using methods from Example 5. (2) The initialclinical validation of the panel used methods from Example 5 and salivasamples from HIV/AIDS patients (n=100) with CD4 counts >500, 200-500 and<200 cells/mm³. The study subjects had stable CD4 counts during 6 monthsbefore enrollment, and also at when tested during the office visit whenthe saliva sample was collected^(68,70-71). The clinical saliva sampleswere collected and processed into smears and protein lysates usingmethods from Example 1. The individual 52 SR markers were measured inthe smears and lysates using optimized ICC and ELISA protocols asdescribed in Example 5. The assays used reference slides and lysateswith normal and increased levels of saliva biomarkers (the N pool and Spool) prepared from normal saliva using methods from Examples 1 and 2.SR marker measurements in the clinical saliva samples were correlatedwith matched CD4 counts (the CD4 count measured during the same officevisit when the saliva was collected). The diagnostic accuracy of the SRmarkers for threshold CD4 counts (>500 and <200 cells/mm³) wasdetermined using the ROC curve analysis as described in Example 5. Aminimal panel of SR markers with the best diagnostic accuracy for thethreshold CD4 counts was selected using methods from Example 5.

(3) Saliva samples were collected from HIV patients (n=100) at 5 timepoints during the initial year of the first-line cART. Prior theenrollment, patients had unsuppressed baseline viral load of ≧500copies/ml and a baseline CD4 count <200 cells/mm³. Benchmarks forsuccessful cART outcome after 9 months (expected in 70-90% of thepatients) were: viral load <50 copies/ml, CD4 count increased >100cells/mm³ above the baseline and no AIDS-defining event or death⁶⁸⁻⁷³.The minimal panel of SR markers produced by the previous study wasmeasured in saliva lysates using multiplexed ELISA as described inExample 5. SR marker measurements were correlated with CD4 count andviral load to determine the prognostic accuracy of SRP markers for cARToutcome. The benchmark for prognostic accuracy was the hazard ratio fromCox proportional hazards models at 95% confidence interval. Thebenchmark for prognostic independence were higher critical chi-squarevalues for Cox models containing SR markers compared to models with CD4count and viral load alone^(69,72). Results of the study were used tooptimize the minimal SR marker panel as outlined in Example 5.

(4) The optimized minimal SR marker panel was used to produce aprototype LFIA device. The prototype was optimized using referencesaliva lysates as described in Example 5. The optimized prototype wastested extensively using clinical saliva samples to demonstratereliability, accuracy, applicability for field use and regulatoryrequirements. The multiplexed ELISA assay was used as a reference assayfor the LFIA. The LFIA device showed actionable levels of AIDS riskbased on threshold CD4 counts: low (CD4 count >500 cells/mm³), moderate(CD4 count 500-200 cells//mm³) or high (CD4 count <200 cells). Thebenefit of identifying a moderate AIDS risk is that it indicates theneed for starting or modifying cART therapy to prevent progression tosevere AIDS risk.

Example 8 SRP Analysis of Post-Traumatic Psychological Stress

Multi-SRP assay of saliva was applied to the study of post-traumaticpsychological stress. Multi-SRP scores were measured in salivary cells(FIG. 8 a). The scores strongly correlated with self-reported healthstatus and provided actionable health care information: normal dailyactivities were possible at baseline and mildly elevated multi-SRPscores, and bed rest was needed at high multi-SRP scores (FIG. 8 b).

FIG. 7: Multi-SRP scores during post-traumatic psychological distress.Saliva samples were collected from a healthy subject at different timepoints before and after psychological trauma. a, Multi-SRP staining ofsaliva cells. Original magnifications: ×200. b, The SRP score wascalculated as the ratio between the average staining intensity across900 saliva cells, and the maximum staining intensity value for salivacells. The staining intensity was quantified using image analysis.Baseline was calculated as the average across multi-SRP scores for sixtime points before the psychological stress. The error bars are standarddeviations. During the psychological distress, multi-SRP scorescorrelated with the functional state. Normal daily activities werepossible till Day 8 when fatigue was reported. Health statusdeteriorated on Day 12 and a bed rest was required due to dizziness andnausea. Normal health status was reported on Day 45 post trauma.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

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What is claimed is:
 1. A device for detecting at least one stressresponse biomarker in a test sample comprising: (a) a disposable modulefor uptake of a test sample and reagent storage, wherein the modulecomprises reagents for assaying for at least one stress responsebiomarker; and (b) a reusable module for signal detection and resultdisplay; wherein the reusable module displays a signal that indicatesthe presence of the at least one stress response biomarker in the testsample.
 2. The device of claim 1 wherein the signal provides a digitalreadout of a percentage above a baseline representing the presence ofthe at least one stress response biomarker in the test sample.
 3. Thedevice of claim 1 wherein the signal provides a visual indicationrepresenting the presence of the at least one stress response biomarkerin the test sample.
 4. The device of claim 3 wherein the visualindication is a color indication.
 5. The device of claim 1 wherein thetest sample is selected from the group containing breath air, saliva,urine, sweat, tears, blood, serum, stool, phlegm, bone marrow,cerebrospinal fluid, seminal fluid, vaginal fluid, amniotic fluid, skin,breast milk, tissue, plant sap, an egg, microbial body, cells suspensionor a combination thereof.
 6. The device of claim 1 wherein the at leastone stress response biomarker is selected from the group consisting ofaldose reductase, apoptosis signal-regulating kinase 1, aquaporin 5,beta-endorphin, betaine GABA transporter, caspase recruitment domainprotein 9, caspase 8, cyclin D, cyclooxygenase 2, cytochrome P450,cytochrome c, c-fos, c-jun, epidermal growth factor receptor, ferritin,glucocorticoid receptor, glucose regulated protein 58, glucose regulatedprotein 75, glutathione S-transferase p, GroEL, heat shock protein25/27, heat shock protein 40, heat shock protein 60, heat shock protein70, heat shock protein 90, heat shock transcription factor-1, hemeoxygenase-1, interleukin 1β, interleukin 6, interleukin 8, interleukin10, interleukin 12, laminin, leptin receptor, matrix metalloproteinase9, metallothionein, Mek-1, Mekk-1, inducible nitric oxide synthase,peripheral benzodiazepine receptor, p38 MAPK, salivary alpha amylase,SAPK, serotonin, serotonin receptor, substance P, superoxide dismutaseMn, superoxide dismutase Cu/Zn, superoxide dismutase EC, transforminggrowth factor β, tumor suppressor p53, and vasoactive intestinalpeptide.
 7. The device of claim 1, wherein the at least one stressbiomarker is associated with dehydration.
 8. The device of claim 1,wherein the at least one stress biomarker is associated with AIDSprogression.
 9. The device of claim 1 wherein the device furthercomprises a means for accessing a database, wherein the databaseprovides a correlation between the presence of the at least one stressbiomarker molecule in the test sample and (i) the presence, absence, orseverity, if present, of a particular disease state; or (ii) thelikelihood that an organism from which the test sample was obtained willcontract or be subject to a particular disease state.
 10. A method fordetecting a condition or disorder associated with a stress response in asubject comprising: detecting an altered level of at least onebiomarkers in an stress response biomarker panel in a sample comprisingsalivary cells from a subject, as compared to a corresponding samplefrom a normal subject, wherein the panel comprises at least twobiomarkers, and wherein further an alteration in the level of biomarkeris indicative of a stress response associated with the condition ordisorder, thereby detecting the condition or disorder in the subject.11. The method of claim 10, wherein the at least one stress responsebiomarker is selected from the group consisting of aldose reductase,apoptosis signal-regulating kinase 1, aquaporin 5, beta-endorphin,betaine GABA transporter, caspase recruitment domain protein 9, caspase8, cyclin D, cyclooxygenase 2, cytochrome P450, cytochrome c, c-fos,c-jun, epidermal growth factor receptor, ferritin, glucocorticoidreceptor, glucose regulated protein 58, glucose regulated protein 75,glutathione S-transferase p, GroEL, heat shock protein 25/27, heat shockprotein 40, heat shock protein 60, heat shock protein 70, heat shockprotein 90, heat shock transcription factor-1, heme oxygenase-1,interleukin 1β, interleukin 6, interleukin 8, interleukin 10,interleukin 12, laminin, leptin receptor, matrix metalloproteinase 9,metallothionein, Mek-1, Mekk-1, inducible nitric oxide synthase,peripheral benzodiazepine receptor, p38 MAPK, salivary alpha amylase,SAPK, serotonin, serotonin receptor, substance P, superoxide dismutaseMn, superoxide dismutase Cu/Zn, superoxide dismutase EC, transforminggrowth factor β, tumor suppressor p53, and vasoactive intestinalpeptide.
 12. The method of claim 10, wherein the at least one stressbiomarker is associated with dehydration.
 13. The method of claim 10,wherein the at least one stress biomarker is associated with AIDSprogression.
 14. The method of claim 10, wherein the levels of the atleast one biomarker are detected by analysis of biomarker protein ornucleic acid in the sample comprising the salivary cells.
 15. The methodof claim 14, wherein the analysis of biomarker protein includesdetection with an antibody.
 16. The method of claim 15, wherein thesalivary cells are lysed prior to analysis with the antibody.
 17. Themethod of claim 16, wherein the analysis is by ELISA.
 18. The method ofclaim 15, wherein the sample comprising the salivary cells is analyzedon microscope slide.
 19. The method of claim 14, wherein the analysis ofbiomarker nucleic acid comprises isolation of salivary cell nucleicacid.
 20. The method of claim 19, wherein the biomarker nucleic acid isdetected in the isolated salivary cell nucleic acid by nucleic acidhybridization or PCR amplification.
 21. A method of processing asalivary cell sample for biomarker analysis comprising: (a) applying asample of saliva or salivary cells to a substrate; (b) fixing the cells;(c) incubating the cells in low pH citrate buffer at 37° C.; (d)contacting the cells with serum; (e) applying a primary antibody foreach of biomarker of a biomarker panel; and (f) detecting the binding ofthe primary antibody using a secondary antibody having a detectablelabel, wherein the label is detected optically using a computerizedimage analysis.
 22. The method of claim 21, wherein the salivary cellsare collected using an oral brush.
 23. The method of claim 21, whereinthe biomarker panel comprises at least one biomarker selected from thegroup consisting of aldose reductase, apoptosis signal-regulating kinase1, aquaporin 5, beta-endorphin, betaine GABA transporter, caspaserecruitment domain protein 9, caspase 8, cyclin D, cyclooxygenase 2,cytochrome P450, cytochrome c, c-fos, c-jun, epidermal growth factorreceptor, ferritin, glucocorticoid receptor, glucose regulated protein58, glucose regulated protein 75, glutathione S-transferase p, GroEL,heat shock protein 25/27, heat shock protein 40, heat shock protein 60,heat shock protein 70, heat shock protein 90, heat shock transcriptionfactor-1, heme oxygenase-1, interleukin 1β, interleukin 6, interleukin8, interleukin 10, interleukin 12, laminin, leptin receptor, matrixmetalloproteinase 9, metallothionein, Mek-1, Mekk-1, inducible nitricoxide synthase, peripheral benzodiazepine receptor, p38 MAPK, salivaryalpha amylase, SAPK, serotonin, serotonin receptor, substance P,superoxide dismutase Mn, superoxide dismutase Cu/Zn, superoxidedismutase EC, transforming growth factor β, tumor suppressor p53, andvasoactive intestinal peptide.
 24. A method for constructing a biomarkerpanel for detecting a stress response in a cultured cell comprising: (a)detecting the level of one or more biomarkers from a panel of biomarkersin cultured cells subjected to a treatment that induces cellular stress;(b) comparing the level of the biomarkers from the treated cells to thelevel of the biomarker from a corresponding sample of cultured cellsthat have not been subjected to the treatment that induces cellularstress, wherein biomarkers having a difference level in the treatedcells as compared to the untreated cells are included in an SR biomarkerpanel for a stress response.
 25. The method of claim 24, wherein thetreatment that induces cellular stress is a stressor selected from thegroup consisting of heat shock, freeze/thaw cycling, hypersalinity,dehydration, and oxidative stress.
 26. The method of claim 24, whereinthe cultured cells are salivary cells, peripheral blood mononuclearcells, or cells from organ cultures of tonsil, skin, gut or lung. 27.The method of claim 24, wherein the cells are animal cells.
 28. Themethod of claim 24, wherein the cells are human cells.
 29. A method fordetecting a condition or disorder associated with a stress response in asubject comprising: detecting an altered level of at least onebiomarkers in an stress response biomarker panel in a sample comprisingsalivary cells from a subject, as compared to a corresponding samplefrom a normal subject, wherein the panel comprises at least twobiomarkers, and wherein further an alteration in the level of biomarkeris indicative of a stress response associated with the condition ordisorder, wherein the altered levels of the at least one or morebiomarkers are detected using a device of claim 1, thereby detecting thecondition or disorder in the subject.